Protein conjugates, methods, vectors, proteins and DNA for producing them, their use, and medicaments and vaccines containing a certain quantity of said protein conjugates

ABSTRACT

The invention relates to protein conjugates, methods, vectors, proteins and DNA for producing them, their use, and medicaments and vaccines containing a certain quantity of said protein conjugates. According to the invention, supramolecular particles are produced that represent one or more different, randomly selectable structural units in a large number on the surface of an individual, approximately spherical protein molecule. Icosahedral lumazine synthases are used as carrier proteins for peptides or proteins. A DNA fragment that encodes a peptide molecule is fused with a DNA fragment that encodes an icosahedral lumazine synthase by molecular-biological methods. Said DNA fragment is inserted into a cloning vector and transformed with an appropriate host strain. A polypeptide is expressed by gene expression. If certain peptide structures are used as the fusion partners, a post-translational change of said structures can be observed in the host strain. The chimeric peptide is purified and chemically modified if necessary. It is possible to produce icosahedral molecules that contain up to 120 different peptide motifs on their surfaces by mixing. The compounds produced lend themselves as auxiliary agents for carrying out analytical methods (ELISA, biosensors) or for producing vaccines.

DESCRIPTION

[0001] Protein conjugates, procedures, vectors, proteins and DNA fortheir preparation, and their utilization as well as pharmaceuticalagents and vaccines containing any of those.

[0002] The invention concerns protein conjugates, procedures, vectors,proteins and DNA for their preparation, and their utilization as well aspharmaceutical agents or vaccines containing any of those. The presentinvention serves for the preparation of supramolecular particles whichdisplay one or several different, arbitrarily selected structural unitsin large numbers on the surface of a single, approximately sphericalprotein molecule.

[0003] Properties of Lumazine Synthase and of Lumazine-Synthase-BasedArtificial Protein Conjugates

[0004] 6,7-Dimethyl-8-ribityllumazine synthase (subsequently designatedlumazine synthase) catalyzes the penultimate step of vitamin B₂biosynthesis in microorganisms and plants. Lumazine synthases fromcertain bacteria (e.g. Escherichia coli, Bacillus subtilis, Aquifexaeolicus) represent highly symmetrical, icosahedral complexes of 60subunits with a molecular weight of approximately 1 MDalton (Bacher andLadenstein, 1991; Bacher et al., 1980; Ladenstein et al., 1986, 1988,1994; Mörtl et al., 1996). X-rays structures of the envelope capsid oflumazine synthase of Bacillus subtilis are known (Ladenstein et al.,1988, 1994; Ritsert et al., 1995). The protein of Bacillus subtilis canbe denatured by the use of urea and can be subsequently renaturated. Theefficacy of renaturation can be enhanced by the addition of a ligand(substrate analog), e.g.5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione or5-nitroso-6-ribitylamino-2,4(1H,3)-pyrimidinedione. The fold of therenaturated protein is identical with the fold of the native lumazinesynthase. In the presence of the said ligand, the lumazine synthase ofBacillus subtilis is stable up to a pH of 10. The environment of theinhibitor molecule is known on basis of the X-ray structure. The bindingsite of this ligand is formed by segments of adjacent monomers (Bacheret al., 1986; Ritsert et al., 1995). This constellation explains thesupportive influence of the ligand during the renaturation of the highmolecular weight protein complexes.

[0005] Lumazine synthases from different microorganisms can be expressedefficiently in recombinant strains of Escherichia coli and Bacillussubtilis. The recombinant proteins can be isolated in high yield.

[0006] The N-terminus as well as the C-terminus are located at thesurface of the icosahedral capsid molecule. For the lumazine synthase ofBacillus subtilis, this was documented for the first time by X-raystructure analysis (Ladenstein et al., 1988). Using DNA synthesis, itwas possible to obtain a gene for the expression of the thermostablelumazine synthase of the hyperthermophilic microorganism, Aquifexaeolicus, which is optimally adapted for the codon usage of Escherichiacoli. The protein can be obtained in high amounts in recombinant form.At a temperature of 80° C., it is stable for at least one week. Theconclusion that the structural relationships are the same in lumazinesynthases of Aquifex aeolicus and Bacillus subtilis can be derived fromthe fact that fusion proteins with an elongation of the C-terminaland/or N-terminal end associate under formation of icosahedral capsidsand from the observation that chimeric proteins consisting of parts oflumazine synthases from Aquifex aeolicus and Bacillus subtilis can beprepared. Consequently, it can be assumed that the quaternary structuresof the enzymes are highly similar.

[0007] Icosahedral lumazine synthases can be functionalyzed at theirsurface by structural units. Oligopeptides or polypeptides whosesegments can be arbitrarily determined are considered preferentially asstructural units (biomolecules). The displayed proteins (conjugatedbiomolecules) are covalently linked with the carrier protein (lumazinesynthase conjugate). A carrier protein is here defined, according to theinvention, as a natural (unmodified) or a modified lumazine synthasewhose primary structure has been modified. In that case, one or severalamino acids can be replaced and/or removed and/or added and/or modified.The conservation of the original catalytic activity of the lumazinesynthase is hereby not required. On the contrary, it is possible to usecatalytically inactive, modified proteins for all applications accordingto the invention.

[0008] The number of conjugated biomolecules on the surface of thecarrier protein can extend over a wide range, whereby, according to theinvention, the surface can be decorated with up to 60 (at one terminus)respectively 120 (at both termini) respectively 180 (at both terminiplus loop insertion) identical or structurally different peptide motifs.Protein subunits on the structural basis of lumazine synthase can alsobe assembled to even larger, approximately spherical particles as wellas tubular structures. These associates can contain well about 60subunits. They do however not possess the strict, geometric regularityof icosahedral, 60-meric lumazine synthase molecules.

[0009] The length of the peptide segments can vary over a wide range,according to the invention, preferentially between 1-500 amino acidresidues, whereby the the peptide motifs can be present in unmodified aswell as modified form.

[0010] Proteins, according to the invention, can also contain one orseveral amino acid analogs, or non-natural amino acids which can beintroduced into the sequence by biological methods (e.g. by suppressortRNA techniques, etc.) or by chemical methods (e.g. by couplingreagents, etc.). Moreover, modifications (e.g. glycosidation etc.) orderivatization (e.g. biotinylation etc.) can be present.

[0011] The respective genetic information for the specification ofpeptide segments which have been artificially introduced in thestructure of lumazine synthase can range from few codons up to severalgenes, depending on whether an oligopeptide, a polypeptide or proteinconsisting of several subunits is intended to be it specified.

[0012] The surface of a lumazine synthase can also be modifiedchemically in such a way that the outer molecular periphery iscovalently linked with a multiplicity of functional regions.

[0013] The production of hetero-oligomeric lumazine synthase conjugatesproceeds via a dissociation step and a subsequent folding/reassociationstep. The proteins which are present in monomeric form afterdenaturation can be mixed ad libitum. Since each of the recombinantsubunits contains one respective constant lumazine synthase part, therenaturation of the lumazine synthase core structure is possible underformation of the natural icosahedral structure.

[0014] Immunological Analysis Methods Based on ELISA Assay Systems

[0015] Antibodies bind with high specificity to certain targetstructures (antigens). Assay methods have been developed based on thedetection of specific antibody-antigen complexes. In order to detectwhether an antibody has bound to its target antigen, severalpossibilities are available. The enzyme-linked immunoassay(enzyme-linked immuno absorbent Assay, ELISA) is one of theseprocedures. In principle, the ELISA can be used for the determination ofany antigen, hapten or antibody; it's predominant application is in thearea of clinical biochemistry. Hereby, it is used to measure, forexample, hematological factors as well as the concentrations of serumproteins such as immunoglobulins, oncofetal proteins and hormones suchas for example insulin. For the diagnosis of infectious diseases,microorganisms such as Candida albicans, rotaviruses, Herpes viruses,HIV or hepatitis B surface antigens are determined in this way.Moreover, immunochemical analysis methods are used for detection ofantibodies for the purpose of diagnosing earlier or current infectiousdiseases (e.g. HIV, hepatitis).

[0016] An ELISA protocol typically comprises the following steps.

[0017] 1. The sample supposed to contain a specific molecule or acertain organism is fixed to a solid support (e.g. microtiter platesmade of plastic).

[0018] 2. Antigens (protein, peptide, hapten-conjugate, etc.) aredetected by specific binding of a specific antibody (primary antibody),which is directed against the respective antigen as described under 1.Hereby, the primary antibody can be labeled per se (e.g. radioactive)and can therefore be localized directly (e.g. by radioautography).Alternatively, the procedure can be continued according to the followingparagraph.

[0019] 3. Frequently, instead of this, a second antibody (secondaryantibody) is added which binds specifically to the primary antibody butnot to the antigen specified under 1. This second antibody is frequentlycoupled chemically with an enzyme (indicator system) which catalyzes theconversion of a colorless substrate into a colored product (e.g.alkaline phosphatase, horseradish peroxidase etc.). The second antibodyis typically directed against the constant segment of the firstantibody. Unbound secondary antibodies are removed by washing.

[0020] 4. Addition of a colorless substrate which is converted into acolored product.

[0021] In the absence of any binding of the primary antibody to theantigens present in the sample, the primary antibody is removed in thefirst washing step. As a consequence, the enzyme-labeled second antibodyalso fails to bind, i.e. the a assay mixture remains colorless. If therespective antigenic structure is available, the primary antibody canbind and the second antibody can bind consecutively. The enzyme coupledto the second antibody catalyzes the color reaction whose product can bedetected easily (e.g. photometrically). The observed enzyme activity isproportional to the content of specific antigen respectively antibody(from Glick, B. Pasternak, J. Molekulare Biotechnologie, SpektrumAkademischer Verlag, 1995, p. 201 ff.).

[0022] In order to perform binding assays, an indicator system (e.g.horseradish peroxidase) is required which permits the visualization ofthe immune reaction which has occurred. The visualization is based onthe stable linkage between the analyzed reactant (antigen or antibody)and an indicator system. As indicators (amplifiers), fluorescent dyes,luminescent dyes, radioactivity, enzymes etc. are used. The indicatorscan be linked covalently or non-covalently to the respective reactant.For example, antigen-antibody binding, biotin-avidin binding or lectinbinding can surve the purpose of stable non covalent linkage betweenindicator and the reaction partner to be detected.

[0023] In case of the direct method, the primary antibody is covalentlylinked to the indicator. The indirect setup circumvents the labeling ofthe primary antibody. The primary antibody is detected by an antibodywhich is labeled with an indicator. This secondary antibody which isobtained from a different animal species binds to all primary antibodiesof any specificity from the first animal species.

[0024] Yet another method of detection consists in the method wherebythree antibodies are used subsequently. The primary antibody fromspecies A is detected by a non-labeled secondary antibody from species Bwhich is present in excess. This is followed by the addition of thetertiary antibody from species A which is linked with an indicator. Thesecondary antibody (bridging antibody) serves as a bridge betweenprimary and tertiary antibody. Through the use of several consecutiveantibodies, the sensitivity can be enhanced.

[0025] Alternatively, the visualization of the bound primary antibodycan occur via other binding systems. The avidin-biotin-complex-bindingis an appropriate system (ABC system). Hereby, the primary or thesecondary antibody must be present in biotinylated form. The indicatorsare likewise biotinylated and are bound to the tetravalent avidin undersaturation of three binding sites. The fourth avidin binding site canbind the biotinylated primary or secondary antibody. Multiplebiotinylation of the indicators used results in very large avidin-enzymecomplexes which increase the sensitivity of the assay system (instead ofavidin, streptavidine can be used). (from Bioanalytik, F. Lottspeich, H.Zorbas, Spektrum Akademischer Verlag, 1998, page 91 ff). With thisprocedure, there is a problem of a further enhancement of sensitivity.

[0026] Signal Amplification Through the Use of a Derivatized MultimericLumazine Synthase in Solution or on an arbitrarily selected surface:

[0027] 1. By interpolation of a biotinylated multimeric lumazinesynthase conjugate (linker protein) between primary antibody andindicator: A lumazine synthase containing up to 60 biotin molecules(e.g. bound through a short linker to the lumazine synthase in order toavoid steric hindrance) on its surface hereby adopts a special positiondue to its spherical, multimeric structure. The binding between antibodyand linker protein respectively between linker protein and indicatoroccurs through the use of an avidin bridge or a streptavidine bridge.Alternatively, avidin- or streptavidine-labeled primary antibodiesrespectively indicators can be used. Linker proteins can be bound to 59of the 60 biotin molecules on the surface of the multimeric linkerprotein, whereby only one biotin molecule is required for bindingbetween primary antibody and linker protein. Through the resultingmultiple binding of enzymes mediating the color reaction, an extremesignal amplification is obtained, where by the signal strength increasesproportional to the antigen concentration.

[0028] 2. Through the use of heterologomeric biotinylated lumazinesynthase conjugates: Through the reassociation, according to theinvention, of different lumazine synthase variants (for examplecombination of 1 to 3 antigen-containing lumazine synthase monomers withup to 59 biotinylated lumazine synthase monomers), a heterooligomericlumazine synthase conjugate is generated which contains a reactant (e.g.antigen) as well as several biotin molecules. To the biotin molecules,streptavidine-mediated (or avidin-mediated oranti-biotin-antibody-mediated) indicator molecules are linked. In anexemplary fashion, two modes of use are described: A) A lumazinesynthase conjugate comprising 1 to 5 short peptides of antigenicallyactive viral or bacterial surface proteins (antigenic determinants) andup to 60 biotin molecules in covalent linkage serves as detectionmolecule for immobilized antibodies which stem from a patient's serum orother fluids. B) Characteristic antibodies against certain infectiousdiseases are harvested with the help of special immobilized epitopes(parts of surface antigens of the respective pathogenic organisms;antigenic determinants) from the respective body fluid. A lumazinesynthase conjugate also containing about 1 to 5 copies of the epitopesdesignated above and up to 60 biotin molecules in covalent linkageserves as detection molecule for the antibodies bound to the immobilizedepitope. In both cases (A and B), a color reaction is obtained throughan arbitrarily selected, streptavidine-coupled enzyme which forms acomplex with the biotinylated lumazine synthase. Through theinterposition of this multiply biotinylated linker protein (lumazinesynthase conjugate) and the multiple binding of color-mediating enzymecaused hereby, a signal amplification is achieved.

[0029] 3. By application of heterooligomeric, non-biotinylated lumazinesynthase conjugates: Through the reassociation of different lumazinesynthase variants, according to the invention, a heterooligomericlumazine synthase conjugate is generated which comprises a reactant(e.g. an antigen which can specifically bind antibodies from a patient'sserum) in one copy as well as epitopes in multiple copies which arerecognized by indicator-labeled antibodies. This again results in signalamplification through multiple binding of antibody-indicator-complexesto the multimeric protein.

[0030] Biosensors

[0031] Classical biochemical methods of analysis such as the immunoassayare based on chemical reaction systems in liquid state. A possiblealternative consists in the application of solid-phase measuring devicesor biosensors. During the last years, the use of biosensors as rapid andsensitive test systems for the detection of diverse materials andmolecules is finding progressively more applications. A biosensorconsists of at least three components: a biological receptor, atransducer and a coupled electronic system. In an immune sensor, thebiological receptor can be an antibody or an antigen coupled to thetransducer in a variety of ways. In both variations, the sensor enablesthe measurement of specifically formed antigen-antibody-complexes.

[0032] For the use as chemical sensors in liquids, e.g. in sera, volumevibrators are especially suitable. They include quartz vibratorslaminated on a specially treated surface (according to the assayprinciple) with antigenic proteins or monoclonal antibodies. Whenalternating current is applied to the quartz, the crystal is excited toelastic vibrations whose amplitude reaches a maximum when the electricalfrequency coincides with a mechanical Eigen-frequency of the respectivequartz. These vibrations can be detected by appropriate measuringdevices. When a quartz crystal laminated with antigens is placed in asolution containing specifically binding antibodies, the latter bind tothe surface, thus modulating the mass of the sensor. The vibronicfrequency is hereby modulated, thus indicating the binding of anantibody. Besides these piezoelectrical immunosensors, efforts are beingmade to develop measuring techniques whose mode of function is similarto potentiometric electrodes resembling those of pH-meters. In thiscase, it is attempted to monitor the modification of the potential whichis generated upon formation of an antigen-antibody-complex on a thinequilibrated layer of silica gel on the surface of the pH-sensitiveglass membrane. Yet another possibility for immunosensor measurementsconsists in the immobilization of proteins (antibodies or antigens) onthe surface of an optical fiber. Interfering waves and surface plasmonsare the optical phenomena which are most frequently used for thispurpose. An interfering wave is formed when light propagating along anoptical fiber is reflected internally. This interfering wave is theelectromagnetic energy arising at the interface of optical fiber andliquid. The energy is absorbed when absorbing molecules are present atthe interface, such that the degree of absorption is proportional to theamount of absorbing material at the interface. The formation ofantigen-antibody complexes, whereby the antigen or the antibody is boundto the fiber surface, can be detected in this way. In the case ofsurface plasmon resonance, a metal-coated glass surface is used asoptical device, whereby an internally totally reflected light beamgenerates an induced electromagnetic surface wave or plasmon. Adetectable surface plasmon resonance arises at a specific angle of theincident light, which depends critically on the refractive index of themedium contacting the metal film. Thus, modifications of this layer,such as those that can be expected after the formation ofantigen-antibody complexes, can be measured.

[0033] Potentiometric immune sensors comprise ion sensitive field effecttransistors. A receptor (the antibody, antigen or other receptor) ishereby attached to the semiconductor gate of the transistor. The bindingof an analyte to the receptor generates a modification of the chargedistribution and thereby an activation of the field effect transistor(from Modrow S., Falke, D., Molekulare Virologie, Spektrum AkademischerVerlag, Heidelberg, Berlin, Oxford, p. 108; Lidell, E. Weeks, I.Antikorper-Techniken, Spektrum Akademischer Verlag, Heidelberg, Berlin,Oxford, pp. 154 ff.). An increase of sensitivity is also desirable incase of these sensor methods.

[0034] Signal amplification through utilization of derivatized,multimeric lumazine synthase molecules on the signal-mediating surface:

[0035] Artificial protein molecules on basis of lumazine synthase canserve as carrier protein, for the construction of a biosensor, e.g. forpresentation of antigenically active catcher peptides for the detectionof antibodies against certain infections. Through the formation of mixedlumazine synthase conjugates, according to the invention, the respectivepeptides can be incorporated into an icosahedral structure, togetherwith a biotin molecule which mediates binding. In this way, up to 59identical or different antigenically active peptides (e.g. domains ofvirus surface proteins) in connection with a biotin molecule, can bepresented on top of an icosahedral molecule. Through the utilization ofseveral different multimeric lumazine synthase conjugates, arepresentative peptide library can be placed on a single sensor. Bindingof the multimeric lumazine synthase conjugate to the surface of atransducer can be enabled, for example, via streptavidine-biotincoupling.

[0036] The sensitivity of such an assay system is significantly enhancedby displaying several antigenic determinants, since not only one singleantibody but several antibodies directed against a specific pathogen canbe detected. Moreover, no well-founded detailed knowledge on specificprotein segments contributing to the binding of antibodies is required,since several proteins of the respective pathogen can be presented onthe sensor with limited effort. Since streptavidine/biotin-coupling canbe used for all epitope presentations, in order to build up a sensor,the same surfaces coated with avidin or streptavidine are requiredthroughout, i.e. the experimental setup does not have to be modified.The respective individual epitope-presenting or biotinylated lumazinesynthase subunits can be easily prepared by recombinant technology. Thishas significant advantages for the development respectively evaluationof diagnostic procedures of this type.

[0037] Through the presence of up to 59 catcher peptides on onemolecule, the surface of the sensor chip (e.g. field effect transistor,plasmon resonance transducer surface etc.) can be increased extremely,thus providing an enormous enhancement of sensitivity. Problems ofstability and specificity are not to be expected upon utilization of athermostable carrier protein and the biotin/streptavidine system.

[0038] In the same way, small molecules can be bound to the surface bysimple chemical coupling. As coupling sites for this purpose, singularexposed reactive amino acids are available on the surface of thespherical protein.

[0039] Principal Structure of a Layer System on Basis of MultimericLumazine Synthase:

[0040] A functionalized lumazine synthase with 60 identically ordifferently modified subunits is linked to a surface (e.g. transducersurface or other arbitrarily selected surface located on a transducer)via an anchor (peptide, fatty acid etc.). The detection sensitivity forbinding of foreign molecules on the surface of the lumazine synthase ishereby enhanced through a high number of functional groups (e.g.epitopes for antibody detection, antibodies for detection of foreignmolecules in solution or other receptors).

[0041] Preparation of Vaccines (In Vitro)

[0042] Vaccinations are conducive to an immunological resistance againstinfectious agents. Vaccines serve predominantly for prevention, i.e.,they should result in the buildup of a protective potential in theimmunized persons whereby it will protect them, upon contact with therespective infectious agent, and thereby protect them from disease. Theinjected orally applied vaccine is conducive to the formation ofantibodies and/or a cellular immune response in the organism.Consequently, upon future exposure, the infectious organism is killed orneutralized with the result that the disease does not break out.

[0043] Infections with bacteria, viruses, fungi and protozoa are a mainfactor of morbidity and mortality worldwide. Through the increasingdevelopment of resistance against virtually all available antibiotics, adeterioration of the morbidity situation is also expected inindustrialized countries. The development of novel vaccines is thereforeof the highest medical significance.

[0044] As vaccines, e.g. attenuated viruses can be applied. Attenuatedviruses resemble infectious agents causing disease, albeit they differfrom them with regard to the virulence behavior; thus they cause only alimited respectively attenuated infection, thereby inducing theformation of neutralizing antibodies and cytotoxic T-cells. Mutations inthe genome of wild type viruses form the molecular basis of attenuation.Attenuated viruses typically generate a very good immune protectionwhich remains intact for several years, but they carry the risk ofbackmutation to the wild type form in the course of the attenuatedinfection.

[0045] Yet another possibility for immunization of humans and animalsconsists in the presentation of antigenically effective parts of surfaceproteins on top of other, non-pathogenic viruses, e.g. plant viruses.The gene fragments specifying an antigenic determinant (e.g. surfaceprotein) of the pathogenically active virus are integrated into thegenome of the non-pathogenic virus (Dalsgaard et al., 1997). The foreignprotein is thereby presented on the surface of the non-pathogenic virus.It is however not possible to integrate DNA fragments above a certainlimiting size into the viral genome. Hence, it is necessary to knowexactly which proteins of the infectious virus are relevant for thegeneration of a protective immune response. A vaccine of this typecannot generate an immune response with the same diversity as thatarising in the course of an infection with the wild type virus or itsattenuated variant. With this type of recombinant vaccine viruses, theimmune response is limited to a selected protein.

[0046] Vaccines consisting of synthetic peptides with a length of 15 to30 amino acids represent a vaccine form which is presently underinvestigation. In this case, individual epitopes of viral proteins whichcause the development of neutralizing antibodies are selected andsynthesized chemically. Solid and detailed knowledge on protein segmentscausing a virus-neutralizing immune response is also required in thiscase. On basis of the high genetic variability of most viruses and thedifferent capacity of individuals to recognize specific protein regionsimmunologically, it would be necessary to combine several epitopes in avaccine based on synthetic peptides. Since there is, beside aluminumhydroxide, no other suitable adjuvant that is generally suited forhumans in order to in enhance the immune response sufficiently, novaccine based on synthetic peptides is hitherto available (from ModrowS., Falke D., Molekulare Virologie, Spektrum Akademischer Verlag,Heidelberg, Berlin, Oxford, p. 87 ff). In contrast to short peptides,high molecular weight molecules such as proteins and carrier-fixedproteins are very well suited as vaccines because they can be appliedwithout the use of auxiliary materials and all the same afford a verygood immunity. The redundant occurrence of antigenic determinants inhigh number, such as in case of viruses or bacteria, on immunogenicmolecules of high molecular weight is favorable for the desired highantigenicity, i.e. a preventive immune response. Lumazine synthase isparticularly suited for this purpose because of its icosahedralstructure. The lumazine synthase consists of at least 60 subunit, i.e.at least 60 equivalent or different antigenic determinants can bepresented on one molecule. The lumazine synthase has a high molecularweight structure and a surface structure which is similar to that ofcertain viruses, i.e. a high antigenicity can be expected. Vaccines ofthis type are free of viral genes and can be prepared with little effortin high yield. Since large viral proteins can be presented, detailed andwell-funded knowledge on protein segments causing a virus neutralizingimmune response is not required.

[0047] The proteins generated by genetic engineering which are thesubject of the present invention are based on the covalent linkage of awild type lumazine synthase or a modified lumazine synthase with partialstructures of viruses, bacteria, fungi, protozoa or toxins. The linkagecan occur at the N-terminus and/or at the C-terminus of the lumazinesynthase. Additionally, the peptides to be presented can be inserted atappropriate sites into the sequence in such a way that they arepresented in the form of a loop on the surface of the multisubunitprotein. It is thereby possible to present a given immunologicaldeterminant in a welldefined high number, e.g. 60-fold or 120-foldaccording to the invention, on top of an icosahedral molecule consistingof 60 subunits with a triangulation number T=1. Moreover, it is alsopossible to prepare associates of high molecular weights comprising morethan 100 subunits (triangulation number T=2 or higher) which are therebyable to present an even larger number of epitopes.

[0048] The association, according to the invention, of subunits withdifferent peptide or protein sequences spliced by genetic engineeringalso offers the possibility for the production of protein moleculeswhich present different antigenic sequences on one given molecule.

[0049] DNA Vaccine

[0050] Since the beginning of the 90's, the possibility to use DNA asvaccine has been under study. The nucleic acids used contain genes orparts of genes of a pathogenic organism specifying an immunogenicprotein. For the development of these vaccines, detailed knowledge onthe immunologically important components is most useful. The genes usedpredominantly specify surface components of a pathogen or parts ofbacterial toxins. They are integrated, together with regulatory elementsfor the control of their expression, into a vector system which isapplied in the form of pure DNA by injection into muscle tissue where itis expressed. Especially in muscle cells, DNA can be detected over longperiods as epsisome, since obviously it is degraded only very slowly.When these respective genes are expressed, the organism can generate ahumoral as well as a cellular immune response. Up to now, this form ofvaccine has been studied in animal models.

[0051] Gene constructs which specify fusion proteins consisting ofprotein components of pathogenic microorganisms and of lumazine synthaseare in principle suitable as DNA vaccines. A DNA vaccine consisting of agene coding for a lumazine synthase (particle-forming component) and aselected gene of the pathogenic agent can be expressed intracellularly,thus affording the production of antigen that can stimulate the immunesystem over long periods. According to current experience with lumazinesynthases from different organisms, the assembly of the icosahedralmolecules in vivo should be possible without auxiliary molecules (cf.chaperonins).

[0052] Oral Vaccines on Plant Basis

[0053] If the immunologically active protein component of the infectionsagent responsible for a protective immune response is known, the genespecifying that peptide can be incorporated into a eukaryotic expressionvector. Subsequent to transformation of plant cells with this DNA,transgenic plants can be obtained which express the respective gene. Theselected protein component can be incorporated by consumption of theplant and can thereupon generate an immune response.

[0054] As a particle forming protein, lumazine synthase to which partsof the immunologically active protein of the pathogenic have been fusedis particularly suitable. By the use of a thermostable, particle-forminglumazine synthase (e.g. from Aquifex aeolicus) as carrier protein, evenboiling-resistant vaccines can be generated.

[0055] Multifunctionally Derivatized Immune Therapeutics on Basis of theMultimeric Lumazine Synthase

[0056] Vaccines are intended to inhibit the multiplication of apathogenic agent and thereby prevent infection. In certain cases it isdifficult to develop a reliable vaccine since the pathogenic organism isnot accessible to antibodies or, as in the case of acquired immunedeficiency (AIDS), too little is known about the pathogenic agent (HIV).The targets of HIV are helper T-cells (helper cells) of the immunesystem, whereby the most important functions of these cells areimpaired. When HIV penetrates into helper cells, the virus is protectedfrom the immunological attack. In the subsequent course of the disease,the infected cell can be destroyed by the production and liberation ofHIV particles. An infected cell can thereby become a “factory” for theproduction of additional virus particles. The most important consequenceof HIV infection is the fact that the immune system can no more provideprotection of ordinary infectious disease. The first step in HIVinfection is the interaction of a 120 kDalton glycoprotein (gp 120) ofthe viral capsid with the CD4 receptor at the surface of the helpercells.

[0057] Antibodies against CD4 block the infection of helper cells underin vitro conditions. The rate of infection is also reduced by an excessof free CD4 protein. A fusion protein comprising parts of the CD4protein and the F_(C) component of an immunoglobulin was developed in anattempt to protect the helper cells as well as to eliminate the virus.The fusion protein is designated CD4 immunoadhesin. The molecule bindsgp120 and blocks HIV; both said activities depend upon the CD4component. The capacity of the fusion protein to bind to cells withF_(C) receptors and the long half life in plasma are due to theimmunoglobulin component. After binding of the immunoadhesin to the freevirus or to an HIV-infected cell, an antibody-dependent, cell-mediatedcytotoxic reaction conducive to the destruction of the virus or theHIV-infected cells is initiated (from Glick, B., Pasternak, J.,Molekulare Biotechnologie, Spektrum Adademischer Verlag, 1995, p. 245).

[0058] The efficiency of that strategy may be improved by the use of amultimeric derivatized lumazine synthase. It is also possible to use afunctionalized lumazine synthase comprising CD4 protein components aswell as F_(C) components. The efficiency should increase considerablysince many of these units rather than one single functional unit arepresent in the molecules.

[0059] Instead of the CD4 component, antibodies (e.g. speciallydeveloped single chain antibodies) directed against a tumor marker (e.g.teratocarcinoma antigen) may be introduced into the multimeric protein,and the functionalized fusion protein may be used for the therapy ofcancer.

[0060] An additional mode of application could consist in thecombination of an antibody against a tumor marker with metallothionein.The multimeric lumazine synthase is hereby decorated with an antitumorantibody and up to 59 metallothionein molecules. The metallothioneinmolecule, in turn, are loaded with radioactive elements (characterizedby short half life time) which are suitable for radiation therapy (e.g.technetium 59). In the course of the therapy, the protein complex bindsto the tumor via it's antibody component, thereby closely apposing thesource of radiation to the tumor tissue. Similar constructs can also beused for diagnostic purposes, e.g. radioactive detection of malignanttumors.

[0061] Utilization of Lumazine Synthase Conjugates for theCharacterization and Purification of Antibodies

[0062] The basis of the foreign peptides is provided by DNA sequencesspecifying a specific epitope. The sequence of the additional peptidesegment can be determined exactly by selection of the DNA sequence.However, it is also possible to incorporate peptide sequencescharacterized by a stochastic amino acid sequence over their entirelength or in partial segments. Multiple stochastic variability can beachieved by the use of synthetic oligonucleotides comprising randomlygenerated sequence segments in order to form representative peptidelibraries. These randomly generated peptides are presented on thesurface of the lumazine synthase and are thus accessible for antibodybinding.

[0063] The resulting lumazine synthase variants (with stochasticvariability of the foreign peptides) can be used, for example, for thecharacterization of antibody binding site. By isolation ofantigen-antibody complexes with subsequent sequencing of the boundpeptide segment (N-terminal Edman sequencing or sequence determinationby mass spectrometry), the selectivity of the binding site of anantibody can be characterized.

[0064] It is also possible to search specifically for antibodiescharacterized by a specific antigen recognition (whereby the antigensequence is known in this case). By application of mixed conjugates,i.e. lumazine synthase conjugates comprising a desired foreign peptide(in multiple form) as well as a biotinylated component (in single form),antibodies can be selectively purified from mixed population. The use ofstreptavidine or avidin coupled to a solid phase is appropriate for thepurpose. The purification, according to the invention, can also beperformed on basis of other affinity materials. The antibodies can beeluted by known standard procedures.

[0065] Solutions for the Described Technical Problems

[0066] The solution of the described technical problems is achieved byproviding the application forms characterized by the patent claims. Theobjective of this invention is the use of lumazine synthase molecules ascarrier proteins for foreign proteins, peptides and/or other moleculesfrom the area of organic chemistry. Moreover, the objective of thisinvention is a method for the selective, recombinant incorporation ofsaid foreign proteins respectively peptides into loops or, according tothe invention, preferentially at the N-terminus and/or at thatC-terminus of lumazine synthases. The method involves an in vivoassociation of different lumazine synthase conjugates by way ofco-expression of the respective genes in one given cell. Moreover, themethod includes the possibility of in vitro reassociation ofindividually designed lumazine synthase conjugates by formation ofspherical particles by way of denaturation/renaturation of monomericsubunits which can be carried out with or without the use of a ligandwhich supports the folding.

[0067] The technology provides lumazine synthase conjugatescharacterized by a peptide accessible to biotinylation (Tucker andGrisshammer, 1996; Schatz, 1993; Cronan, 1990) at the C-terminus.Moreover, the technology provides an artificial lumazine synthasemolecule characterized by a well accessible basic amino acid (lysine) atthe C-terminus. Moreover, the technology provides a lumazine synthasemolecule characterized by a well accessible cystein molecule at theC-terminus. Both variants are suitable for chemical coupling of organicmolecules. Coupling can be achieved by the generation of an amide bondor a disulfide bond between protein and coupling component. Chemicalcoupling according to the amide principle can also occur at the lysinresidues which are naturally present on the surface of lumazine synthasemolecules.

[0068] Moreover, the technology provides a thermostable, icosahedrallumazine synthase (from Aquifex aeolicus) which is suitable as carrierprotein for the preparation of particularly stable lumazine synthaseconjugates.

[0069] The procedure for the preparation of lumazine synthase conjugatesinvolves the following steps:

[0070] I. Preparation of Fusion Vectors

[0071] A) Preparation of a DNA containing a gene for a lumazine synthase(e.g. by isolation from an organism, by PCR amplification with naturallyoccurring RNA or DNA as template or by DNA synthesis).

[0072] B) Introduction of suitable restriction sites for the laterinsertion of foreign DNA into the lumazine synthase gene; adaptation ofthe lumazine synthase sequence to particular requirements using knownmutagenesis methods based on molecular biological and biochemicalmethods; insertion of the DNA into a cloning vector by application ofknown molecular biology methodology. (Alternatively, the DNA coding forthe foreign peptide can be fused directly with the lumazine synthasegene using the polymerase chain reaction and synthetic oligonucleotides,whereby II.D must be granted.

[0073] C) Transformation of host cells with the resulting plasmid

[0074] D) Selection of transformants by use of antibiotics or otherselection procedures

[0075] E) Analysis of transformants by means of molecular biology andbiochemistry methods such as restriction mapping, sequencing,measurement of enzyme activity etc.

[0076] II. Insertion of a DNA Specifying a Foreign Peptide

[0077] A) Cloning of the foreign DNA by means of molecular biologymethodology or preparation of a DNA by use of chemical synthesismethodology

[0078] B) Analysis of the DNA using molecular biology technology

[0079] C) Preparation of the DNA specifying the foreign peptidedesignated for fusion

[0080] D) Insertion of the prepared DNA at the 5′ and/or the 3′ endand/or into a loop region of the lumazine synthase gene in the vectorprepared under I. in order to fuse the foreign gene with the lumazinesynthase gene. The cloning must occur in such a way that all used genesegments are incorporated in the correct reading frame in order toarrange for all fused gene segments to be jointly translated into afusion protein.

[0081] E) Transformation of host cells with the resulting plasmid

[0082] F) Selection of transformants using antibiotics or otherselection procedures

[0083] G) Analysis of transformants by means of molecular biology orbiochemistry methodology such as restriction mapping, sequencing,measuring of enzymatic activity etc.

[0084] III. Expression and Purification of the Hybrid Polypeptides

[0085] A) Fermentation of the host strain with the artificial fusion DNAusing known microbiological methods

[0086] B) Expression of the fused artificial DNA in the transformed hostcells as chimeric protein. The expression of the artificial DNA caninvolve a purposeful post-translational modification of the chimericprotein in vivo, e.g. phosphorylation, glycosidation, biotinylation etc.

[0087] C) Preparation of a cell extract with the fusion polypeptide

[0088] D) Purification of the fusion protein by means of chromatographicor other methods

[0089] E) If required: Solubilization and in vitro folding(renaturation)

[0090] F) If required: Chemical modification of the surface of lumazinesynthase variants

[0091] G) If required: In vitro association under combination ofdifferent lumazine synthase variants

[0092] Additional Explanation of the Methodology:

[0093] The application of the present invention can involve a multitudeof different vectors. Extra-chromosomal (episomal) vectors (e.g.plasmids), integration vectors (e.g. lambda vectors), Agrobacteriumtumefaciens-based vectors designed for plants (e.g. Ti-plasmid).According to the invention, plasmid vectors are preferred. The plasmidsused can have been isolated from natural sources or can be preparedsynthetically. The selected plasmid should be compatible with therespective host strain. Therefore, it should have a replication originsuitable for the respective host strain. Moreover, the capacity of thevector should be sufficient for the used lumazine synthase variant aswell as the fused foreign peptide. Moreover, singular restriction sitesfor the cloning of DNA fragments are required. The plasmid vector shouldhave suitable features such as a resistance gene in order to enableappropriate selection procedures. The selection is necessary in order todistinguish host cells with and without plasmid.

[0094] If Escherichia coli is selected as host strain, vectors using apromoters sequences from bacteria phage T5 or T 7, an operator sequence,preferably the operator sequence of the Escherichia coli lactose operon(lacO), a cloning site with several singular restriction sites forrestriction endonucleases and an efficient terminator sequence arepreferred according to the invention. Moreover, the vector should have areplication origin providing for a high copy number of theextrachromosomal DNA in the host cells.

[0095] Prokaryotic expression systems are in general well-suited for therecombinant production of protein conjugates according to the invention.In certain cases, however, post-translational modifications may berequired which cannot be introduced in prokaryotic organisms. Forexample, eukaryotic proteins cannot be glycosidated or phosphorylated.Therefore, eukaryotic foreign proteins (fused to lumazine synthase)requiring such a post-translational modification are expressedpreferentially under the control of a strong promoter (e.g. AOX1) inlower eukaryots (e.g. Pichia pastoris) or under the control of apromoter specific for mammalian cells (e.g. rat preproinsulin promoter)in mammalian cells (e.g. COS7 monkey kidney cells) or under the controlof a promotor (e.g. polyhedrin promoter) specific for insect cells(Baculovirus, Autographa californica). The respective factors usedshould be compatible to the said host strains.

[0096] For production of oral vaccines on basis of plants, it is forexample possible to use vector systems on basis of the Ti plasmid ofAgrobacterium tumefaciens. As an alternative to gene transfer in plants(e.g. monocotyl plant such as rice, wheat, maize etc.), physical methods(e.g. the gene gun technology, biolistic technology) can be used.

[0097] Naturally occurring proteins as well as proteins which do notoccur in nature can be fused to the carrier protein (lumazine synthase).As sources of DNA, it is for example possible to use viruses,prokaryotic (eubacteria, archaea) and eukaryotic organisms (plants,animals). The DNA selected for fusion can also be prepared syntheticallyusing established technology. Moreover, DNA can be prepared on basis ofmRNA using reverse transcriptase.

[0098] The plasmid vectors obtained by recombinant technology are usedfor the transformation of host cells. Well characterized bacterial cellsare preferred according to the invention. The host cells can also beeukaryotic cells. The host strains used should provide the enzymesystems required for expression of the fused polypeptide. Transformationtechniques are well known in the field. Specific procedures aredescribed in Maniatis et al. (1982). Subsequent to the transformation,transformants are analyzed. The plasmids are isolated and characterizedby molecular biology methods such as restriction analysis and DNAsequencing.

[0099] The expression of the cloned DNA sequence in a prokaryotic oreukaryotic host cell can be performed by well-known technology.Cultivation of transformed host cells, according to the invention, forthe preparation of recombinant fusion proteins proceeds under conditionswhich are favorable for the expression of the DNA sequence. Celldisruption subsequent to gene expression can be performed by all methodsgenerally accepted for that purpose. Disrupted cells are separated intoa soluble and an insoluble fraction by known separation procedures.

[0100] If the fusion protein is present in the insoluble fraction in theform of inclusion bodies, the pellet obtained by centrifugation iswashed and subsequently dissolved by the addition of a solubilizer.Solubilization is preferentially performed in presence of reducingagents. Insoluble components are removed by known procedures. Accordingto the invention, the renaturation step can be performed in presence ofa stabilizing agent (5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione).

[0101] Purification of the fusion proteins can be performed using knownchromatographic or other biochemical methods.

[0102] Covalent coupling of molecules by chemical methods is enabled orfacilitated by the introduction of a reactive amino acid usingrecombinant technology, preferably a lysine and/or cystein residueaccording to the invention, which is coupled to a flexible peptidelinker. According to the invention, coupling can be performed by severaldifferent methods. The following examples are given specifically: a)Bismid esters are well soluble in water and can be coupled with the εamino group of a lysine residue under mild reaction conditions (pH7.0-pH 10.0). The resulting amide bond is stable. Lumazine synthasesactivated in this way can be used for coupling with other peptides. b)Carbodiimides belong to a group of compounds described by the generalformula R—N═C═N—R′. The residues R respectively R′ can be aliphatic oraromatic moieties. Carbodiimides react preferentially with the ε aminogroup of lysine. c) m-Maleimido-benzoyl-N-hydroxysuccinimide ester (MBS)is a well studied heterobifunctional reactant. In neutral aqueoussolution, MBS reacts initially via an acetylation type reaction underformation of an activated N-hydroxysuccinimide ester. A second peptidecan then be bound via addition of a thiol residue to the double bond ofthe ester. d) N-Succinmidyl-3-(2-pyridyldithio)-propionate (SPDP) is aheterobifunctional reagent which can react under mild conditions withamino groups of the target proteins. The 2-pyridyldisulfide structurecan then react with aliphatic thiols or a cystein residue of anadditional peptide by thiol disulfide exchange reaction. The couplingreaction can proceed in the pH range of 5-9 and the reaction progresscan be monitored photometrically. No reactions with other functionalgroups are known.

[0103] The preparation, according to the invention, of lumazine synthaseconjugates by in vitro reassociation proceeds via a dissociation stepand a subsequent folding/reassociation step. The dissociation can occurby a treatment with denaturating agents, e.g. urea or guanidinechloride, by modification of the pH value, by heat treatment or by otherprocedures. The monomeric chimeric proteins which are present afterdenaturation comprise a constant region of a lumazine synthase(respectively a modified lumazine synthase) and a variable region (afused peptide which can be selected arbitrarily). Subsequently, themonomeric subunits can be mixed arbitrarily. Since each respectiverecombinant subunit comprises a respective constant lumazine synthasepart, renaturation of the lumazine synthase core structure underformation of the natural icosahedral structure is possible. Therenaturation can proceed in presence of a stabilizing agent(preferentially 5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione).

[0104] The in vivo combination of different lumazine synthase variantsproceeds by way of co-expression of the respective gene coding for therespective fusion polypeptide. Here by, the respective genes can belocated on the chromosomal DNA of the host strain and/or on one orseveral plasmid vectors. By modulation of the expression of the lumazinesynthase variants to be associated in vivo, a specific ratio ofcombinatorial variants can be established.

LEGENDS TO FIGURES

[0105]FIG. 1 gives a schematic representation of an ELISA protocol forthe determination of a specific antigen or a specific antibody. Theantigen is bound to the microtiter plate. The enzyme (E) is coupled tothe secondary antibody. The colorless substrate is converted to acolored product by the enzyme (E).

[0106]FIG. 2 describes the detection of an antigen by way of abiotin-labeled primary antibody. A lumazine synthase conjugate(amplifying linker molecule) comprising up to 60 covalently bound biotinmolecules is linked to a biotinylated primary antibody via astreptavidine or avidin bridge (SA). The color reaction occurs by way ofan arbitrarily selected streptavidine coupled enzyme (E) which forms acomplex with the biotinylated lumazine synthase. Through theinterposition of a 60-fold biotinylated linker protein (lumazinesynthase conjugate) and the multiple binding mediated thereby of a colorreaction mediating enzyme, an extreme signal enhancement is obtained,whereby the signal strength is proportional to the antigenconcentration.

[0107]FIG. 3 describes the use of a lumazine synthase mixed conjugatefor the diagnosis of infectious disease.

[0108] A) A lumazine synthase molecule carrying 1-5 short peptides fromantigenically active viral or bacterial surface proteins (antigenicdeterminants, epitops) and up to 60 biotin molecules in covalent linkageserves as detection molecule for immobilized antibodies which stem froma patient's serum or other fluid.

[0109] B) Characteristic antibodies directed against specific infectiousdiseases are harvested by means of special immobilized epitopes (partsof surface proteins of the respective pathogenic organisms; antigenicdeterminants) from the respective body fluid. A lumazine synthasemolecule which also contains 1-5 copies of the said epitopes and up to60 biotin molecules in covalent linkage serves as detector molecule forthese hereby immobilized antibodies.

[0110] A color reaction is obtained in both cases by an arbitrarilyselected streptavidine coupled enzyme (E) which forms a complex with thebiotinylated lumazine synthase. Through the interposition of a multiplybiotinylated linker protein (lumazine synthase conjugate) and themultiple binding of color reaction mediating enzyme mediated hereby, asignal amplification is obtained.

[0111] Non-bound antibodies are removed in a first washing step. If nobinding to the immobilized epitopes occurs, the complex of lumazinesynthase conjugate and antibody is not formed. Excessive lumazinesynthase conjugate is removed in a second washing step, such that theassay mixture remains colorless.

[0112]FIG. 4 describes a schematic representation of an experimentalsetup for the purification of antibodies characterized by a specificantigen recognition. A lumazine synthase conjugate comprising a desiredforeign peptide (in multiple form) as well as a biotinylated moiety (insingular form) is bound to immobilized streptavidine via its biotinmoiety. The streptavidine molecules are coupled to a solid phase. Themixed antibody population is applied to a column of immobilizedstreptavidine (or is mixed with streptavidine material), whereby theantibodies with the desired specificity bind to the foreign peptidemoiety of the lumazine synthase conjugate. The washing process of thestreptavidine lumazine synthase conjugate/antibody complex and thesubsequent elution of the specific antibodies occurs by known standardmethods.

[0113]FIG. 5 shows a systematic representation of the structure of abiosensor which can consist, in principle, of three parts: 1. Thebiological receptor, 2. The transducer unit, 3. The integratedelectronic unit. The biological receptor can be linked to the transducerin various ways.

[0114]FIG. 6 shows a functionalized lumazine synthase with 60 identicalrespectively differently modified subunits bound to a surface (forexample transducer surface, membrane, other surface etc.) via an anchor(peptide, fatty acid, other functional group etc.). The detectionsensitivity for binding of foreign molecules at the surface of thelumazine synthase is enhanced by the large number of functional groups.(for example epitopes for antibody recognition, antibodies for detectionof foreign molecules in solution or other receptors).

[0115]FIG. 7 schematically shows a possible structure of a field effecttransistor under inclusion of a multimeric functionalized lumazinesynthase. A modification of the surface charge of the gate electroderesulting from the binding of a foreign molecule to the surface of thelumazine synthase hereby modulates the flux of current through the fieldeffect transistor.

[0116]FIG. 8 shows a sequence comparison of lumazine synthases from thefollowing organisms: 1. Mycobacterium avium; 2. Mycobacteriumtuberculosis; 3. Corynebacterium ammoniagenes; 4. Chlorobium tepidum; 5.Aquifex aeolicus; 6. Thermotoga maritima; 7. Bacillus subtilis; 8.Bacillus amyloliquefaciens; 9. A. pleuropneumoniae; 10. Streptococcuspneumoniae; 11. Staphylococcus aureus; 12. Vibrio cholerae; 13.Photobacterium phosporeum; 14. S. putrefaciens; 15. Photobacteriumleiognathi; 16. Shigella flexneri; 17. Escherichia coli; 18. Haemophilusinfluenzae; 19. Dehalospirillum multivorans; 20. Helicobacter pylori;21. Deinococcus radiodurans; 22. Synechocystis sp., 23. Porphyromonasgingivalis; 24. Arabidopsis thaliana; 25. Methanococcus jannaschii; 26.Archaeoglobus fulgidus; 27. Methanobacterium thermoautotrophicum, 28.Chlamydia trachomatis; 29. Saccharomyces cerevisiae; 30. Brucellaabortus. The protein sequences were obtained by translation of thecognate DNA sequences. The set of sequences shown was obtained bydatabase search using the search algorithm according Altschul et al.(1997) and the sequence of lumazine synthase of Bacillus subtilis asearch motif.

[0117]FIG. 9 shows a top view of the pentameric subunit of theicosahedral lumazine synthase of Bacillus subtilis. The ligand5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione binds to the contactsite between two monomeric subunits (Ladenstein et al., 1988, 1994;Ritsert et al., 1995).

[0118]FIG. 10 shows a model of the icosahedral lumazine synthase ofBacillus subtilis. One out of 12 pentameric subunits is emphasized bythe use of different gray tones. The N-terminus as well as theC-terminus are located at the surface and are readily accessible.

[0119]FIG. 11 shows the expression vectors used in the applicationexamples. SD, ribosomal binding site; MCS, cloning cassette withsingular cutting sites; t₀, t₁, terminator sequences; (cat), inactivegene for chloramphenicol acetyl transferase (shifted reading frame);(Δcat), inactive gene for chloramphenicol acetyl transferase (deletion);restriction sites are indicated by letters: B, BamHI; E, EcoRI; H,HindIII; N, NcoI; P, PstI; S, SalI. Translation start in vector pNCO 113at position 113 and at position 233 for vector p602/-CAT.

[0120]FIG. 12 describes the 1. PCR for introduction of a mutation using,as an example, the introduction of a mutation of the amino acid cysteinin position 93 against serine in the gene for lumazine synthase ofBacillus subtilis. In the first step of the directed mutagenesis,initially, two separate PCR reactions were performed with theoligonucleotides pairs PNCO-M1/C93S and PNCO-M2/RibH-3 and theexpression plasmid pNCO-BS-Lusy as template. Fragment A contains thedesired mutation and an intact recognition sequence for the restrictionnuclease EcoRI. Fragment B represents the entire, but non-mutagenizedribH gene (lumazine synthase of Bacillus subtilis). In this fragment,the 5′ restriction cloning site is deleted. (R: ribosomal binding site)

[0121]FIG. 13 describes the 2. PCR for introduction of a mutation. Inthe second step of the mutagenesis, the mutation to be introduced whichis now still at the 3′ end of the PCR-generated gene fragment isintroduced into the entire gene by overlapping elongation.

[0122]FIG. 14 describes the 3. PCR for introduction of a mutation. The3. PCR serves the amplification of the elongated codon strand offragment A.

[0123] In the FIGS. 15-24, 26-28 and 30 and 31, the sequence of therespective lumazine synthases is emphasized by bold type. The linkerregions are underlined. Recognition sequences for the respectiverestriction endonucleases are italicized and underlined. Fused sequencesrespectively amino acids which are not part of the linker sequence aremarked by punctuated underlining. The amino acid sequence is given inthe one letter code.

[0124]FIG. 15 shows the structure of the vector pNCO-N-BS-LuSy for thefusion of foreign proteins to the N-terminus of lumazine synthase.

[0125]FIG. 16 shows the structure of the vector pNCO-C-BS-LuSy for thefusion of foreign proteins to the C-terminus of lumazine synthase.

[0126]FIG. 17 shows the structure of the vector pNCO-BS-LuSy-EC-DHFR.

[0127]FIG. 18 shows the structure of the vector pNCO-N-VP2-BS-LuSy inthe region of the N-terminus.

[0128]FIG. 19 shows the structure of the vector pNCO-C-VP2-BS-LuSy inthe region of the C-terminus.

[0129]FIG. 20 shows the structure of the vector pNCO-C-Biotag-BS-LuSy inthe region of the C-terminus.

[0130]FIG. 21 shows the structure of the vector pNCO-Lys165-BS-LuSy inthe region of the C-terminus.

[0131]FIG. 22 shows the structure of the vector pNCO-Cys167-BS-LuSy inthe region of the C-terminus.

[0132]FIG. 23 shows the structure of the vector pFLAG-MAC-BS-LuSy in theregion of the N-terminus.

[0133]FIG. 24 shows the structure of the vector pNCO-C-His6-BS-LuSy inthe region of the C-terminus.

[0134]FIG. 25 shows the construction of the thermostable lumazinesynthase of Aquifex aeolicus (Deckert et al., 1998) using 11 syntheticoligonukleotides (AQUI-1 tos AQUI-11) and 6 steps of polymerase chainreaction.

[0135]FIG. 26 shows the coupling of an artificial peptide with a lengthof 13 amino acids, which is accessible to in vivo biotinylation, to theC-terminus of the thermostable lumazine synthase of Aquifex aeolicus.(The peptide is bound to the C-terminus of the carrier protein by alinker of 3 alanine residues)

[0136]FIG. 27 shows the coupling of an artificial peptide with thelength of 13 amino acids, which is accessible to in vivo biotinylation,to the C-terminus of the thermostable lumazine synthase of Aquifexaeolicus by a linker of 6 histidine and 3 alanine residues.

[0137]FIG. 28 shows the coupling of an artificial peptide with a lengthof 13 amino acids, which is accessible to in vivo biotinylation, to theC-terminus of the thermostable lumazine synthase of Aquifex aeolicus bya linker consisting of 6 histidine residues and the sequenceGly-Gly-Ser-Gly-Ala-Ala-Ala

[0138]FIG. 29 shows the production of a chimeric protein consisting of apart of the lumazine synthase of Bacillus subtilis and a part of thethermostable lumazine synthase of Aquifex aeolicus

[0139]FIG. 30 shows the 5′ region of the vector pNCO-AA-BglII-LuSyrespectively the vector pNCO-AA-BglII-LuSy-(BamHI) for the fusion offoreign genes to the 5′ end of lumazine synthase of Aquifex aeolicus.The recognition sequence for the singular restriction nuclease BglIInewly introduced into the sequence is marked.

[0140]FIG. 31 shows the 3′ region of vector pNCO-AA-BgIII-LuSyrespectively pNCOAA-BglII-LuSy-(BamHI) for fusion of foreign genes tothe 3′ end of lumazine synthase of Aqufex aeolicus. A peptide with thesequence GSVDLQPSLIS is fused to the C-terminus of the sequence.

[0141] The describes DNA sequence protocols illustrate the structure ofthe plasmids shown in the examples. In the sequence protocols, therecognition sequences of the respective restriction endonucleases usedare underlined and italicized; the expressed fusion proteins are shownin bold type, and linker sequences are shown is punctuated underlining;exceptions in the formatting are indicated.

[0142] SEQ ID No.1 shows the DNA sequence of the expression vectorpNCO113 (vector for expression of genes in Escherichia coli; Stüber etal., 1990).

[0143] SEQ ID No.2 shows the DNA sequence of the expression vectorp602/-CAT (shuttle vector for expression of genes in Escherichia coliand Bacillus subtilis; Henner, 1990; LeGrice, 1990).

[0144] SEQ ID No.3 shows the DNA sequence of the expression plasmidpNCO-BS-LuSy (expression plasmid with an unmodified lumazine synthase ofBacillus subtilis for expression in Escherichia coli).

[0145] SEQ ID No.4 shows the DNA sequence of the expression plasmidp602-BS-LuSy (expression plasmid with an unmodified lumazine synthase ofBacillus subtilis for expression in Escherichia coli and Bacillussubtilis).

[0146] SEQ ID No.5 shows the DNA sequence of the expression plasmidpNCO-BS-LuSy-C93S (expression plasmid with a modified lumazine synthasevariant, whereby the amino acid cystein in position 93 was exchanged bythe amino acid serin).

[0147] SEQ ID No.6 shows the DNA sequence of the expression plasmidpNCO-BS-LuSy-C139S (expression plasmid with a modified lumazine synthasevariant, whereby the amino acid cystein in position 139 was exchanged bythe amino acid serin).

[0148] SEQ ID No.7 shows the DNA sequence of the expression plasmidpNCO-BS-LuSy-C93/139S (expression plasmid with a modified lumazinesynthase variant, whereby the amino acid cystein in positions 93 and 139was exchanged by the amino acid serin).

[0149] SEQ ID No.8 shows the DNA sequence of the expression vectorpNCO-N-BS-LuSy for the fusion of foreign peptides to the N-terminus ofthe lumazine synthase of Bacillus subtilis.

[0150] SEQ ID No.9 shows the DNA sequence of the expression vectorpneCO-C-BS-LuSy for the fusion of foreign peptides to the C-terminus ofthe lumazine synthase of Bacillus subtilis.

[0151] SEQ ID No.10 shows the DNA sequence of the expression vectorpNCO-EC-DHFR-BS-LuSy (expression plasmid for expression of a fusionprotein consisting of dihydrofolate reductase of Escherichia coli andthe lumazine synthase of Bacillus subtilis, whereby the dihydrofolatereductase is fused to the N-terminus of lumazine synthase).

[0152] SEQ ID No.11 shows the DNA sequence of the expression vectorpNCO-EC-MBP-BS-LuSy. (expression plasmid for expression of a fusionprotein comprising maltose binding protein of Escherichia coli and thelumazine synthase of Bacillus subtilis, whereby the maltose bindingprotein is fused to the N-terminus of lumazine synthase). SEQ ID No.12shows the DNA sequence of the expression vector pNCO-BS-LuSy-EC-DHFR.The linker sequence between the lumazine synthase and the dihydrofolatereductase is underlined in punctuated lines. (Expression plasmid forexpression of a fusion protein consisting of the dihydrofolate reductaseof Escherichia coli and the lumazine synthase of Bacillus subtiliswhereby the dihydrofolate reductase is fused to the C-terminus of thelumazine synthase).

[0153] SEQ ID No.13 shows the DNA sequence of the expression vectorpNCO-N-VP2-BS-LuSy. (Expression plasmid for expression of a fusionprotein consisting of the VP2-domain of the “Mink enteritis virus” andthe lumazine synthase of Bacillus subtilis, whereby the VP2-domain islocated at the N-terminus; the pristine start codon of the lumazinesynthase is underlined).

[0154] SEQ ID No.14 shows the DNA sequence of the expression vectorpNCO-C-VP2-BS-LuSy. (Expression plasmid for expression of a fusionprotein consisting of the VP2-domain of the “Mink enteritis virus” andthe lumazine synthase of Bacillus subtilis, whereby the VP2-domain islocated at the C-terminus).

[0155] SEQ ID No.15 shows the DNA sequence of the expression vectorpNCO-N/C-VP2-BS-LuSy. (Expression plasmid for expression of a fusionprotein consisting of the VP2-domain of the “Mink enteritis virus” andthe lumazine synthase of Bacillus subitlis, whereby the VP2-domain islocated at the N-terminus as well as at the C-terminus; the pristinestart codon of the lumazine synthase is underlined).

[0156] SEQ ID No.16 shows the DNA sequence of the expression vectorpNCO-C-Biotag-BS-LuSy. (Expression plasmid for expression of a fusionprotein consisting of a peptide consisting of 13 amino acids which issusceptible to biotinylation in vivo, and of the lumazine synthase ofBacillus subitlis, whereby the fused peptide is located at theC-terminus).

[0157] SEQ ID No.17 shows the DNA sequence of the expression vectorpNCO-Lys165-BS-LuSy. (Expression plasmid for expression of a modifiedlumazine synthase of Bacillus subitlis, whereby the C-terminus has beenelongated and ends with a lysine residue; the codon for lysine (AAA) isunderlined).

[0158] SEQ ID No.18 shows the DNA sequence of the expression vectorpNCO-Cys167-BS-LuSy. (Expression plasmid for expression of a modifiedlumazine synthase of Bacillus subitlis, whereby the C-terminus has beenelongated and ends with a cystein residue; the codon for cystein (TGC)is underlined).

[0159] SEQ ID No.19 shows the DNA sequence of the expression vectorpFLAG-MAC-BS-LuSy. (Expression plasmid for expression of a fusionprotein comprising an epitope which consists of 12 amino acids that canbe recognized by a monoclonal antibody, as well as the lumazine synthaseof Bacillus subtilis, whereby the fused peptide is located at theN-terminus; the pristine start codon of the lumazine synthase isunderlined).

[0160] SEQ ID No.20 shows the DNA sequence of the expression vectorpNCO-C-His6-BS-LuSy. (Expression plasmid for expression of a fusionpeptide comprising a peptide with the length of six amino acids(6×histidine) and the lumazine synthase of Bacillus subtilis, wherebythe fused peptide is located at the C-terminus; the peptide isunderlined).

[0161] SEQ ID No.21 shows the DNA sequence of the expression vectorpNCO-AA-LuSy. (Expression plasmid for expression of the unmodified,thermostable lumazine synthase of Aquifex aeolicus; the DNA sequence hasbeen adapted to the codon usage of Escherichia coli; the DNA has beensynthesized in its entirety).

[0162] SEQ ID No.22 shows the DNA sequence of the expression vectorpNCO-C-Biotag-AA-LuSy. (Expression plasmid for expression of a fusionprotein comprising a peptide with the length of 13 amino acids which issusceptible to biotinylation, and the lumazine synthase of Aquifexaeolicus, whereby the fused peptide is located at the C-terminus; thepeptide is connected to the C-terminus of the carrier protein by alinker of 3 alanine residues).

[0163] SEQ ID No.23 shows the DNA sequence of the expression vectorpNCO-His6-C-Biotag-AA-Lusy. (Expression plasmid for the expression ofthe lumazine synthase of Aquifex aeolicus with a C-terminal peptidewhich is susceptible to in vivo biotinylation and which is coupled via alinker of 6 histidine and 3 alanine residues).

[0164] SEQ ID No.24 shows the DNA sequence of the expression vectorpNCO-His6-GLY2-SER-GLY-C-Biotag-AA-LuSy. (Expression plasmid for theexpression of the lumazine synthase of Aquifex aeolicus with aC-temrinal peptide which is susceptible to in vivo biotinylation andwhich is coupled via a linker with the amino acid sequenceHHHHHHGGSGAAA).

[0165] SEQ ID No.25 shows the DNA sequence of the expression vectorpNCO-BS-LuSy-AgeI-AA-LuSy. (Expression plasmid for expression of achimeric protein consisting a part of lumazine synthase of Bacillussubtilis and a part of the thermostable lumazine synthase of Aquifexaeolicus; the Bacillus subtilis lumazine synthase part is shown in boldtype, the Aquifex aeolicus lumazine synthase part is double underlined).

[0166] SEQ ID No.26 shows the DNA sequence of the expression vectorpNCO-AA-BglII-LuSy (Vector for fusion of foreign peptides to theN-terminus respectively to the 5′ end of the thermostable lumazinesynthase of Aquifex aeolicus using the restriction endonuclease BglII).

[0167] SEQ ID No.27 shows the DNA sequence of the expression vectorpNCO-AA-LuSy-(BamHI). (Vector for fusion of foreign peptides to theC-terminus respectively to the 3′ end of the thermostable lumazinesynthase of Aquifex aeolicus using the restriction endonuclease BamHI).

[0168] SEQ ID No.28 shows the DNA sequence of the expression vectorpNCO-AA-BglII-LuSy-(BamHI). (Vector for fusion of foreign peptides tothe N-terminus and the C-terminus respectively to the 5′ and 3′ ends ofthe thermostable lumazine synthase of Aquifex aeolicus using therestriction endonuclease BamHI).

EXAMPLES Example 1

[0169] Heterologous Expression of the Gene (ribH) Coding for theIumazine Synthase from Bacillus subtilis in Escherichia coli XL1 Cells

[0170] A) The gene coding for the lumazine synthase from Bacillussubtilis was amplified using the oligonucleotide RibH-1 (5′ gag gag aaatta acc atg aat atc ata caa gga aat tta g 3′) as forward primer, whichwas at his 3′-end identical to the 5′-end of the ribH gene and whichcoded for an optimized ribosome binding site at his 5′-end. As reverseprimer the oligonucleotide RibH-2 (5′ tat tat gga tcc cca tgg tta ttcgaa aga acg gtt taa gtt tg 3′) was used, which was at his 3′-endidentical to the 3′-end of the ribH gene and which introduced arecognition site for the restriction endonuclease BamHI (G*GATCC) inclose distance to the stop codon. The plasmid pRF2 (Perkins et al.,1991) was used as template for the PCR (Mullis et al., 1986).

[0171] 10 μl PCR-buffer (75 mM Tris/HCl, pH 9.0; 20 mM (NH₄)₂SO₄; 0.01%(w/v) Tween 20)

[0172] 6 μl Mg²⁺[1.5 mM]

[0173] 8 μl dNTP's [each 200 μM]

[0174] 1 μl RibH-1 [0.5 μM]

[0175] 1 μl RibH-2 [0.5 μM]

[0176] 1 μl pRF2 [10 ng]

[0177] 1 μl Goldstar-Taq-Polymerase [0.5 U] (Eurogentec, Seraing,Belgien)

[0178] 72 μl H₂O_(bidest)

[0179] PCR cycle protocol (GeneAmp® PCR System 2400; Perkin Elmer):

[0180] 1. 5.0 min 95° C.

[0181] 2. 0.5 min 94° C.

[0182] 3. 0.5 min 50° C.

[0183] 4. 0.8 min 72° C.

[0184] 5. 7.0 min 72° C.

[0185] 6. ∞ 4° C.

[0186] Steps 2.-4. were repeated 20 times.

[0187] B) The PCR mixture was analyzed and separated on an agarose gel,the DNA was visualized using ethidium bromide and UV light and the DNAfragment with a length of 498 bp was isolated from the gel. The DNAfragment was purified using the Geneclean II-Kit from Bio101 (San Diego,Calif., USA) according to the manufacture's instructions. In the laststep the DNA was eluted using 30 μl bidest. water and a incubtiontemperature of 45° C. for 15 min. The concentration of the DNA wasmeasured by fluorescense spectroscopy using the intercalation dyebisbenzimide H 33258 (Höchst, Frankfurt, Germany). The measuring wascarried out by an excitation of 365 nm, and emission of 458 nm. Theblank was measured with 2 ml of TNE buffer (100 mM Tris/HCl pH 7.4, 10mM EDTA, 1 M NaCl) which contained 0.1 μg/ml H 33258. 2 μl plasmid DNAwith known concentration was used as DNA standard for the calibration.

[0188] C) 10 ng of the purified DNA from B) served as a template for a2. PCR. using the oligonucleotide EcoRI-RBS-1 (5′ ata ata gaa ttc attaaa gag gag aaa tta acc atg 3′), which was identical to the 5′-end ofprimer RibH-1 and which extended the ribosome binding site in5′-direction. In close 5′ contact to the ribosome binding site, arecognition site for the endonuclease EcoRI (G*AATTC) was introducedinto the DNA fragment. The oligonucleotide RibH-2 was used as reverseprimer.

[0189] 10 μl PCR-buffer

[0190] 6 μl Mg²⁺[1.5 mM]

[0191] 8 μl dNTP's [each 200 μM]

[0192] 1 μl EcoRI-RBS [0.5 μM]

[0193] 1 μl RibH-2 [0.5 μM]

[0194] 1 μl DNA from B) [10 ng]

[0195] 1 μl Goldstar-Taq-Polymerase [0.5 U] (Eurogentec, Seraing,Belgien)

[0196] 72 μl H₂O_(bidest)

[0197] PCR cycle protocol (GeneAmp® PCR System 2400; Perkin Elmer):

[0198] 1. 5.0 min 95° C.

[0199] 2. 0.5 min 94° C.

[0200] 3. 0.5 min 50° C.

[0201] 4. 0.8 min 72° C.

[0202] 5. 7.0 min 72° C.

[0203] 6. ∞ 4° C.

[0204] Steps 2.-4. were repeated 20 times.

[0205] D) The PCR mixture was analyzed and separated on agarose gel anda DNA fragment with a length of 516 bp was isolated according to B).

[0206] E) The isolated DNA-fragment was digested using the restrictionendonucleases EcoRI and BamHI.

[0207] 30.0 μl DNA from D)

[0208] 2.5 μl EcoRI [62.5 U]

[0209] 3.0 μl BamHI [60 U]

[0210] 24.0 μl OPAU (10×; 500 mM pottasium acetate; 100 mM magnesiumacetate; 100 mM tris-acetate, pH 7.5)

[0211] 60.5 μl H₂O_(bidest)

[0212] The enzymes were purchased from Pharmacia Biotech (Freiburg,Germany). The mixture was incubated for 180 min at 37° C. After theincubation the mixture was purified according to B) and used in aligation protocol.

[0213] F) The expression vector was digested using the restrictionendonucleases EcoRI and BamHI.

[0214] 25.0 μl pNCO113 [5 μg]

[0215] 2.5 μl EcoRI [62.5 U]

[0216] 3.0 μl BamHI [60 U]

[0217] 24.0 μl OPAU (10×)

[0218] 65.5 μl H₂O_(bidest)

[0219] The enzymes were purchased from Pharmacia Biotech (Freiburg,Germany). The mixture was incubated for 180 min at 37° C. After theincubation the mixture was purified according to B) and used in aligation protocol.

[0220] G) The DNA fragments resulting from E) and F) were ligated in amolecular relation of 3 to 1 (Sgamarella, 1979).

[0221] 1 μl expression vector from F) [50 fmol]

[0222] 2 μl DNA-fragment from E) [150 fmol]

[0223] 4 μl H₂O_(bidest)

[0224] mix, 10 min/55° C., 5 min on ice

[0225] 2 μl T₄-Puffer (5×; 250 mM tris/HCl, pH 7.6; 50 mM MgCl₂; 5 mMATP; 5 mM DTT;

[0226] 25% (w/v) polyethylene glycole-8000)

[0227] 1 μl T₄-Ligase [1 U] (Gibco BRL, Eggenstein, Germany)

[0228] The mixture was incubated at 4° C. overnight yielding the plasmidpNCO-BS-LuSy.

[0229] H) Preparation of electrocompetent Escherichia coli XL1-cells(Dower et al., 1988) and electroporation: 1 liter LB-medium (10 g/lpeptone; 5 g/l yeast extract; 5 g/l NaCl) was inoculated with 10 ml of aXL1 cell suspension which was grown overnight at 28° C. The cell culturewas then incubated in a incubator under shaking at 37° C. At an opticaldensity (600 nm) of 0.5 to 0.7 the culture was placed on ice for 15 min.Cells were harvested by centrifugation (Sorvall-GS-3-Rotor, 2300 rpm, 4°C., 15 min). The cell pellet was suspended in 1 liter sterile glycerolsolution (10% in water, w/w) and the mixture was centrifuged again usingthe same conditions. The resulting pellet was then washed with 500 mlglycerol solution, centrifuged and at least washed with 20 ml glycerolsolution and centrifuged again. After the last centrifugation step thepellet was suspended in 2-3 ml of glycerol solution and placed on ice(electrocompetent cells). The electroporation tube (0,1 cm) and the tubeholder were cooled on ice for 15 min. 40 μl of electrocompetent cellswere mixed with 1-2 μl of the ligation mixture from G) in a precooled1.5 ml cap and after that transferred to the precooled electroporationtube. The electroporation was carried out in a electroporation devicefrom Biorad (Munich, Germany). Conditions: 25 μF, 1.8 kV, 200 Ω. Afterthe pulse the suspension was mixed with 1 ml of SOC medium (2% peptone;0.5% yeast extract; 10 mM NaCl; 2.5 mM KCl; 10 mM MgCl₂; 10 mM MgSO₄; 20mM glucose). The transformation mixture was then incubated for 1 h at37° C. in a shaker. After this step 20 μl and 200 μl aliquots wereplated on LB-Amp-agar-plates (21 g/l Agar; 10 g/l peptone; 5 g/l yeastextract; 5 μl NaCl; 150 mg/l ampicilline) and incubated overnight at 37°C. resulting in the expression strain XL1-pNCO-BS-LuSy.

[0230] I) A plasmid (pNCO-BS-LuSy) form H) was isolated using the methoddescribed by Birnboim und Doly (1979). Cells from a 100 ml overnightculture were suspended in 4 ml of buffer S1 (50 mM tris/HCl, 10 mM EDTA,100 μg RnaseA/ml, pH 8.0) and then 4 ml of buffer S2 (200 mM NaOH, 1%SDS) was added. After gentle shaking of the suspension and 5 minincubation at room temperature, 4 ml of buffer S3 (2.6 M KAc, pH 5.2)was added. The resulting mixture was incubated for 20 min on ice. Aftercentrifugation (Sorvall-SS34-Rotor, 17000 rpm, 4° C., 30 min) thesupernatant was placed on a Nucleobond® AX100 column (Macherey undNagel, Düren) which was equilibrated with 2 ml of buffer N2 (0.9 M KCl;100 mM tris-phosphate, pH 6.3; 15% (v/v) ethanol). The column was washedusing 8 ml buffer N3 (1.3 M KCl, pH 6.3). After that the DNA was elutedusing 2 ml buffer N5 (1.3 M KCl, pH 8.0). The DNA was precipitated using1.4 ml isopropanole and the DNA-pellet was washed twice with icecoldethanol (70% in water, (v/v)). After that the pellet was dried in avacuum centrifuge and the resulting DNA-pellet was solved in 200 μlbidest. water.

[0231] J) The isolated plasmid (pNCO-BS-LuSy) from I) was sequencedusing the chain termination method from Sanger et al. (1971). Thesequencing mixture contained 1 μg plasmid-DNA from I), 10 pmolsequencing primer Seq-1 (5′ gtg agc gga taa caa ttt cac aca g 3′), 10 μlterminator Premix™ (dNTP's, ddNTP's, labeled ddNTP's undTaq-DNA-polymerase) from ABI (Weiterstadt, Germany) and bidest. water toa endvolume of 21 μl. The reaction was carried out in a GeneAmpPCRSystem 2400 device from Perkin Elmer (Norwalk, Conn., USA).

[0232] PCR cycle protocol:

[0233] 15 s/96° C.

[0234] 15 s/50° C.

[0235] 4 min/72° C.

[0236] The PCR steps were repeated 20 times.

[0237] In a following step 80 μl bidest. water was added and the mixturewas shaked out two times using 100 μlphenole/chloroform/amylalcohole-mix (25:24:1) from ABI (Weiterstadt,Germany). The DNA was pecipitated with 300 μl ethanol containing 10 μl 3M Na-acetate. The suspension was then centrifuged (14000 rpm, RT, 30min) and the resulting pellet was washed with ethanol (70%, v/v, icecooled) and dried in a vacuum centrifuge. The DNA was then solved in asolution containing 1 μl 50 mM EDTA, pH 8.0 and 5 μl formamide. The DNAwas incubated 2 min at 95° C. and then cooled on ice. 1.5 μl of thissolution was placed on a 4.75% polyacrylamide-sequencing gel.Preparation of the polyacrylamide gel: 13.3 ml ofUltraPureSequagel™Sequencing-System-conzentrate from NationalDiagnostics (Atlanta, Ga., USA) was mixed with 49.7 mlUltraPureSequagel™Sequencing-System-Diluent and deionized with AmberliteMB-1. The suspension was filtrated (0.2 μm) and 7 mlUltraPureSequagel™Sequencing-System-buffer was added. After deairing,210 μl ammonium peroxodisulfate-solution (10%, w/w) and 25 μl TEMED wereadded. The mixture was placed in a gel tray. The developing of the gelwas carried out in TBE buffer (1 M Tris-Base, pH 8.3; 0.85 M Boron acid;10 mM EDTA) using a Prism™377-DNA-Sequencer from Perkin-Elmer-ABI(Weiterstadt, Germany).

[0238] K) Expression strains containing an expression plasmid from I)were fermented in 25 ml LB-AMP-medium (10 g/l peptone; 5 g/l yeastextract; 5 g/l NaCl; 150 mg/l ampicilline). The culture was inoculatedwith 500 μl of an overnight culture from H) (relation: 1:50 (v/v)).After an optical density (600 nm) of 0.7 the expression was induced bythe addition of IPTG (isopropyl-β-D-thiogalactopyranoside) resulting ina final concentration of 2 mM. At an additional incubation of 5 h, cellswere harvested by centrifugation (5000 rpm 4° C., 15 min). The pelletwas washed with 5 ml 0.9% NaCl (w/v) (20% of the culture volume) andstored at −20° C.

[0239] L) Cells from K) were thawed and lysed using an ultrasonic devicefrom Branson SONIC Power Company (Branson-Sonifier B-12A, Branson SONICPower Company, Dunbury, Conn., USA). The cell pellet from K) suspendedin 800 μl lysis-buffer (50 mM K-phosphate, pH 7.0; 10 mM EDTA; 10 mMNa₂SO₃; 0.3 mM PMSF; 0.02% Na-azide) and incubated for 10 min on ice.The cell suspension was then lysed using the ultrasonic device (onepulse for 8 sec and level 4.5). The suspension was then cooled on icefor 5 min and lysed under the same conditions for a second time. Afterthe second sonication the suspension was centrifuged(Eppendorff-centrifuge; 15000 rpm, 4° C., 15 min) and the supernatant(crude lysate) was used for the following steps.

[0240] M) To check the expression level and the molecular weight of themonomeric subunit of the expressed lumazine synthase a SDS gelelectrophoresis (SDS-PAGE) according to Laemmli (1970) was carried out.As a matter of routine gels with 4% acrylamide in the collecting gel and16% acrylamide in the separating gel were prepared (acrylamidestocksolution: 38.8% (w/v) acrylamide; 1.2% (w/v) N,N′-methylenebisacrylamide). The crude lysate from L) has been diluted 1:2, 1:5 and1:10 with sample buffer (20% glycerol; 4% 2-mercapto ethanol; 4% (w/v)SDS; 0.05% bromphenolblue) and boiled for 15 min. After cooling down thesamples were centrifuged (15000 rpm, 5 min, 4° C.) and 8 μl of the clearsupernatant were used for the SDS-PAGE. As molecular weight standard weused Dalton Mark VII-L from Sigma (Deisenhofen, Germany) containingmarker proteins with molecular weights of 66, 44, 36, 29, 24, 20 und 14kDa (Standard proteins). The electrophoresis was carried out by aconstant voltage of 20 mV. After the development of the gel it wasstained with coomassie blue dye (40% methanol; 10% acetic acid; 0.2%(w/v) coomassie Blue R 250). To remove the dye out of the polyacrylamide gel (not out of the protein) a dye removing solution was used (40%methanol; 10% acetic acid; 50% water). In the crude lysate of the strainXL1-pNCO-BS-LuSy a protein band with a molecular weight of circa 16 kDacould be observed. This protein band couldn't be observed in aEscherichia coli strain without the expression plasmid pNCO-BS-LuSy. Theobserved protein band corresponded to circa 10% of the total solubleproteins of the Escherichia coli strain.

[0241] N) To check the enzymatic function of the protein an enzyme assayusing the native substrates5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidindione andL-3,4-dihydroxy-2-butanone-4-phosphate; Bacher et al., 1997) was carriedout. The assay mixture contained 100 mM K-phosphate-buffer pH 7.0, 4 mMEDTA, 0.6 mM 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidindione (PYR;obtained by catalytic reduction of5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidindione), 2 mM DTT, 1 mML-3,4-dihydroxy-2-butanone-4-phosphate (DHP) and crude lysate from L).In a first step the mixture was incubated (without PYR) for 3 min at 37°C. Afterwards the reaction was started by addition of PYR and incubatedat 37° C. After several time intervals (2, 5 and 10 min) aliquots of themixture were removed and the reaction in those aliquots was stoped bythe addition of TCA (15% in water; (w/v)) and centrifuged (15000 rpm, 5min, room temperature). The quantity of the product of the enzymereaction (6,7-dimethyl-8-ribityllumazine) was checked by HPLC (column:reverse phase Nucleosil 10C₁₈ (4×250 mm); excitation: 407 nm; emission:487 nm; elution buffer: 7% methanol, 30 mM formic acid). As standardchemical synthesized 6,7-dimethyl-8-ribityllumazine was used. One unit(1 U) of the enzyme 6,7-dimethyl-8-ribityllumazine synthase catalyzedthe formation of 1 nmol 6,7-dimethyl-8-ribityllumazine per hour at 37°C. In the crude lysate of the strain XL1-pNCO-BS-LuSy a volume activityof 15600 U/ml could be measured. After determination of the totalprotein concentration (13 mg/ml) of the crude lysate according to O) aspecific activity of 1200 U/mg could be calculated.

[0242] O) The determination of the protein concentration in the crudelysate was carried out using a modified variant of the Bradford-method(Read and Northcote, 1981; Compton and Jones, 1985). Thereactive-reagent contained 0.1 g Serva Blue G, 100 ml 16 M phosphoricacid and 47 ml ethanol. The solution was filtrated and stored in thedark at 4° C. The crude lysate was diluted 50-fold with bradford-buffer(2.0 g Na₂HPO₄, 0.6 g KH₂PO₄, 7.0 g NaCl, 0.2 g Na-azide per literwater; (w/v)). 50 μl of the diluted solution was mixed with 950 μl ofreactive-reagent and incubated at room temperature for 15 min.Afterwards the extinction of the mixture was determined at 595 nm. Eachmeasurement was carried out three times and summarized to a mean value.As a blank a solution containing 50 μl bradford-buffer and 950 μlreactive-reagent was used. The blank was handled under the sameconditions. Each sample was measured three times. For the calibrationbovine serum albumin with known concentration was used and the proteinconcentration of the crude lysate calculated on the basis of thecalibration curve.

[0243] P) To carry out negative staining experiments on a electronmicroscope grids coated with formvar/carbon were used. Circa 10 μlprotein solution (≈1 mg/ml) were placed on the grid and incubated for 1min at room temperature. The surplus protein solution which wasn'tadsorbed to the grid was removed after the incubation period. Afterwardsthe grid was incubated with uranyl acetate (30 sec; 2% in water) andwashed with water. This procedure was repeated 2-3 times. Subsequent thegrid was dried and placed in the grid holder of the electron microscope(JEM-100CX, Jeol, Japan). Negative staining shots showed hollowspherical particles with an outer diameter of circa 15 nm and an innerdiameter of circa 5 nm.

[0244] Q) The western blot analysis was carried out according to amethod from Sambrook et al. (1989). Starting from a denaturingSDS-polyacrylamide gel (16%) proteins were transfered on a PVDF membraneby electro blotting (constant current: 40 mA, 2 h). After thetransference of the proteins, the membrane was rinsed inantibody-washing-solution-A (20 mM Tris, pH 7.4; 150 mM NaCl; 3 mM KCl;0.05% Tween 20). Afterwards the membrane was incubated inantibody-washing-solution-B (antibody-washing-solution-A containing 3%skimmed milk powder) for 1 h at room temperature. Subsequent themembrane was incubated overnight in 5 ml antibody-washing-solution-C(antibody-washing-solution-A containing 1% skimmed milk powder)containing 10 μl Anti-sRFS solution (primary antibody; rabbit crudeserum with polyclonal antibodies against lumazine synthase from Bacillussubtilis; diluted 1:10 in antibody-washing-solution-C). Afterwards themembrane was washed 3 times using 5 ml antibody-washing-solution-A.Subsequent the membrane was incubated in 5 mlantibody-washing-solution-C containing 20 μl secondary antibodyconjugate (Anti-rabbit-IgG-HRP-conjugate in 50% glycerole; Sigma,Munich, Germany). Afterwards the membrane was washed 3 times using 5 mlantibody-washing-solution-A. The visualization of the lumazine synthasewas carried out using the substrates for the horse radish peroxidase3,3′-diaminobenzidine (6 mg in 10 ml antibody-washing-solution-A) and 10μl perhydrole (30%). The lumazine synthase could be detected on themembrane as a single band with a molecular weight of circa 16 kDa.

[0245] R) The isolation of the lumazine synthase from the Escherichiacoli strain XL 1-pNCO-BS-LuSy was carried out in two steps. Thefermentation of the cells was carried out according to K), however in avolume of 1 liter. After washing the cells in 0.9% NaCl (w/v; 20% of theculture volume) the pellet was suspended in 32 ml lysis-buffer (L)) andcooled on ice for 10 min. Afterwards the cells were lysed using aultrasonic device from Branson SONIC Power Company (Branson-SonifierB-12A, Branson SONIC Power Company, Dunbury, Conn., USA; 15 pulses atlevel 5). The suspension was then cooled on ice for 5 min and lysedunder the same conditions for a second, third and forth time. After theforth sonication the suspension was centrifuged (Sorvall SS34-Rotor;15000 rpm, 4° C., 15 min) and the supernatant was applied to anionexchange column (DEAE-Cellulose DE52; 2×15 cm, Whatman Ltd., Maidstone,GB) equilibrated with buffer A (50 mM K-phosphate, 10 mM EDTA, 10 mMNa-sulfite, 0.02% Na-azide, pH 7.0). The column was rinsed using 100 mlbuffer A. After that the column was developed using a salt gradient from50 mM phosphate (buffer A) to 1 M phosphate (buffer B: 1 M K-phosphate,10 MM EDTA, 10 mM Na-sulfite, 0.02% Na-azide, pH 7.0; gradient profile:101 ml to 200 ml 15% buffer B; 201 ml to 500 ml 18% buffer B; 501 ml to650 ml 100% buffer B) with a flow rate of 1 ml/min. The lumazinesynthase could be eluted at a salt concentration of 250 mM phosphate.The fractions were checked for lumazine synthase activity according toN). Enzymatic active fractions were collected and dialysed againstbuffer A in a volume ratio of 1:1000 (18 h, 4° C.). The dialysed proteinsolution was concentrated using an ultra centrifuge (Beckman LE 70 withrotor 70Ti; 32000 rpm, 18 h, 4° C.). The concentrated protein solution(75% pure) was applied to gel filtration column which had beenequilibrated with buffer A (Sepharose-6B, 2×180 cm, Pharmacia Biotech,Freiburg, Germany). The column was developed using buffer A (flow rateof 0.5 ml/min). The fractions were checked for lumazine synthaseactivity according to N). Enzymatic active fractions were collected andconcentrated using an ultracentrifuge (Beckman LE 70 with rotor 70Ti;32000 rpm, 18 h, 4° C.).

[0246] S) The purity check was carried out according to M) (SDS-PAGE)whereby only one band could be observed at a molecular weight of circa16 kDa. The enzymatic activity was measured according to N), the proteinconcentration was determined according to O). Using these data aspecific activity of 12400 U/mg could be calculated. Negative stainingshots according to P) showed hollow spherical particles with an outerdiameter of 15 nm and an inner diameter of 5 nm.

[0247] T) To check the quarternary structure of the pure lumazinesynthase a native gel electrophoresis using a 3.5% poly acryl amide gelwas carried out. The gel was prepared using 5.7 ml acrylamide stocksolution (38.8% (w/v) acrylamide; 1.2% (w/v)N,N′-methylenbisacrylamide), 46 ml gel buffer (0.2 M Na-phosphate, pH7.2), 13 ml H₂O_(bidest), 300 μl ammoium peroxodisulfate solution (10%(w/v) in water), 65 μl TEMED (N,N,N′,N′-tetramethylethylene diamine) and5 mg bromo phenole blue were mixed and applied to a gel preparing device(Pharmacia Biotech, Freiburg, Germany) which contained a GelBond® PAGFilm (FMC Bioproducts, Rockland, Me., USA) and polymerized overnight atroom temperature. 20 μl of the pure protein solution (concentration:0.2-1 mg/ml) were applied to the gel. The electrophoresis was carriedout in gel buffer at constant 100 mA under temperature control (10° C.).The staining was carried out according to M). The purified recombinantlumazine synthase was observed as a distinct single band on the gel andthe behaviour was comparable to a lumazine sample which had beenisolated from a wild type Bacillus subtilis strain.

[0248] U) To check the quarternary structure respectively the structuralhomogenity of the pure protein a sedimentation analysis on an analyticalultra centrifuge (Optima XLA with rotor AN60 Ti, Beckman Instruments,Munich, Germany) was carried out (Laue et al., 1992). The protein wascentrifuged at 45000 rpm and every 5 min the radial change in theabsorption (280 nm) was measured and the movement of the proteindetermined. The recombinant lumazine synthase sedimented as a singlehomogenous band, i.e. there was only one molecular species present inthe analyzed sample. A sedimentation constant S_(20,w of) 26.3 S couldbe calculated.

[0249] V) For a precise determination of the native molecular weight ofthe pure lumazine synthase an analytical ultracentrifugation was carriedout using an equilibrium sedimentation protocol. For the radius relateddetermination of the protein concentration the absorption was measuredat 280 μm. Samples with an absorption of 0.3 at 280 nm were used. 150 μlof this protein solution was filled into the sample sector of acentrifuge cell and 15 μl oil (Fluorochemical FC 43, Beckman, Munich,Germany) was added. The reference sector was filled with 200 μl buffer A(R). The centrifugation was carried out at 3000 rpm til an equilibriumstate was reached. The data were calculated using the softwareXLA-Data-Analysis from Beckman Instruments. The partial specific volumeof the protein was estimated according to Cohn and Edsall (1943) basedon the partial specific volumes of each amino acid residue of theprotein and temperatur corrections. The purified recombinant lumazinesynthase showed a molecular weight of 925 kDa at 4° C. (60 mer).

Example 2

[0250] Homologous Expression of the Gene (ribH) Coding for the LumazineSynthase from Bacillus subtilis in Bacillus subtilis BR151-pBL1 Cells

[0251] In comparision to the heterologous expression of the ribH gene inEscherichia coli (Example 1) the homologous expression of the ribH genein Bacillus subtilis is more efficient relating to the yield of theexpressed recombinant protein.

[0252] A) Analogous Example 1 A) to E), excepting that oligonucletideEcoRI-RBS-2 (5′ ata ata gaa ttc att aaa gag gag aaa tta act atg 3′) wasused instead oligonucleotide EcoRI-RBS-1.

[0253] B) The expression vector p602/-CAT was cut analogous to Example 1F). The resulting DNA-Fragment, with a length of 5269 bp, was purifiedaccording to Example 1 B) and used in a ligation protocol.

[0254] C) The ligation protocol was carried out analogous to Example 1G) yielding the expression plasmid p602-BS-LuSy.

[0255] D) The transformation of Escherichia coli XL1-celles was carriedout analogous to Example 1H), excepting that LB-KAN-agar plates (21 g/lagar; 10 g/l peptone; 5 g/l yeast extrakt; 5 g/l NaCl; 15 mg/lkanamycine) were used instead of LB-AMP-agar plates (the vectorp602/-CAT includes a kanamycine resistence gene).

[0256] E) The isolation of the resulting expression plasmid was carriedout analogous to Example 1 I), excepting that LB-KAN liquid medium (10g/l peptone; 5 g/l yeast extrakt; 5 g/l NaCl; 15 mg/l kanamycine) wasused instead of LB-AMP liquid medium.

[0257] F) DNA-sequencing was carried out analogous to Example 1 J),excepting that oligonucleotide Seq-2 (5′ gta taa tag att caa att gtg agegg 3′) was used instead of oligonucleotide Seq-1.

[0258] G) The fermentation of the expression strain was carried outanalogous to Example 1 K), excepting that LB-KAN liquid medium was used.

[0259] H) Cell lysis was carried out analogous to Example 1 L).

[0260] I) The SDS-PAGE (protocol analogous Example 1 M)) of the crudelysate of the Escherichia coli expression strain XL1-p602-BS-LuSy showeda distinct overexpressed protein band at a molecular weight of circa 16kDa. The expression rate of the recombinant lumazine synthase wasestimated to 30% related to the total soluble cell proteins of therecombinant strain.

[0261] J) The enzymatic activity was measured according to Example 1 N),the protein concentration was determined according to Example 1 O).Using these data a specific activity of circa 3700 U/mg could becalculated in the crude lysate.

[0262] K) The preparation of electrocompetent Bacillus subtilis-cellswas carried out using a modified protocol according to Brigidi et al.(1989). 500 ml LB-ERY-liquid medium (10 g/l peptone; 5 g/l yeastextract; 5 g/l NaCl; 15 mg/l erythromycine) was inoculated with 5 ml ofa BR151[pBL1] cell suspension which was grown overnight at 32° C. Thecell culture was then incubated in a incubator at 32° C. At an opticaldensity (578 nm) of 0.6 the culture was placed on ice for 30 min. Cellswere harvested by centrifugation (Sorvall-GS-3-Rotor, 2300 rpm, 4° C.,15 min). The cell pellet was suspended in 300 ml 1 mM HEPES buffer (1 mM(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] in water, pH7.0) and the mixture was centrifuged again using the same conditions.The resulting pellet was washed twice with 200 ml PEB buffer (272 mMsuccrose; 1 mM MgCl₂; 7 mM K-phosphate, pH 7.4) and centrufuged againusing the same conditions. After the last centrifugation step the pelletwas suspended in 16 ml PEB buffer and placed on ice (electrocompetentcells).The electroporation tube (0.4 cm) and the tube holder was cooledon ice for 15 min. 800 μl of electrocompetent cells were mixed with500-1500 ng of plasmid-DNA (p602-BS-LuSy from E)) in a precooled cap andincubated on ice for 10 min. After transferance into the precooledelectroporation tube the electroporation was carried out in aelectroporation device from Biorad (Munich, Germany). Conditions: 25 μF,2.5 kV. After the pulse the suspension was mixed with 6 ml LB-ERY-mediumand incubated at 32° C. for 2 h (transformation mixture A). Subsequently25 ml of LB-ERY-KAN-medium (10 g/l peptone; 5 g/l yeast extract; 5 g/lNaCl; 15 mg/l erythromycine; 15 mg/l kanamycine) were mixed with 1 ml ofthe transformation mixture A and incubated for 4-8 h in a shaker at 32°C. (transformation mixture B). After this step 20 μl and 200 μl aliquotswere removed from transformation mixture B after 2, 4, 6 and 8 h, platedon LB-ERY-KAN-Agar-plates (21 g/l Agar; 10 g/l peptone; 5 g/l yeastextract; 5 μl NaCl; 15 mg/l erythromycine; 15 mg/ml kanamycine) andincubated overnight at 32° C. resulting in the expression strainBR151-pBL1-p602-BS-LuSy. In parallel to that 100 μl, 200 μl and 400 μlof transformation mixture A were plated on LB-ERY-KAN-Agar-plates andincubated overnight at 32° C.

[0263] L) The resulting transformants were checked for the presence ofthe plasmid p602-BS-LuSy using PCR. The PCR was carried out analogous toExample 1 A), excepting that the PCR mixture was prepared without addingtemplate DNA. Aliquots of the PCR mixture were inoculated with cellsfrom fresh transformants using sterile toothpicks yielding a fragmentwith 498 bp. After this step LB-ERY-KAN-agar plates were inoculated withthe specific toothpick (copy of the checked clone) and incubated at 32°C. overnight.

[0264] M) The fermentation of the cells was carried out usingLB-ERY-KAN-liquid medium (10 g/l peptone; 5 g/l yeast extract; 5 g/lNaCl; 15 mg/l erythromycine; 15 mg/l kanamycine) analogous to Example 1K), excepting that a temperature of 32° C. instead of 37° C. was used.The cells were incubated for additional 18 h after induction (additionof IPTG).

[0265] N) The cells were lysed analogous to Example 1 L).

[0266] O) The SDS-PAGE (protocol analogous Example 1 M)) of the crudelysate of the Bacillus subtilis BR151-pBL1-p602-BS-LuSy strain showed adistinct overexpressed protein band at a molecular weight of circa 16kDa. The expression rate of the recombinant lumazine synthase wasestimated to 40-50% related to the total soluble cell proteins.

[0267] P) The enzymatic activity was measured according to Example 1 N),the protein concentration was determined according to Example 1 O).Using these data a specific activity of circa 4000 U/mg could becalculated in the crude lysate of the expression strainBR151-pBL1-p602-BS-LuSy.

[0268] Q) Negative staining experiments were carried out analogous toExample 1 P) yielding comparable results.

[0269] R) The western blot analysis was carried out analogous to Example1 Q) yielding comparable results.

[0270] S) The recombinant lumazine synthase from the Bacillus subtilisstrain BR151-pBL1-p602-BS-LuSy could be isolated in pure form using onesingle column (Sepharose-6B, 2×180 cm, Pharmacia Biotech, Freiburg,Germany). The fermentation was carried out analogous to M) exceptingthat 1 liter medium was used. The cells were lysed analogous to Example1 R) in 32 ml lysis buffer, excepting that 30 mg lysozyme was added tothe lysis buffer. In a first step the suspension was incubated at 37° C.for 1 h. In a second step the cells were lysed using a ultrasonic deviceand centrifuged analogous to Example 1 R). Subsequent the supernatantwas filtrated (0.22 μm).). The filtrated protein solution was applied tothe gel filtration column which had been equilibrated with buffer Aanalogous Example 1 R). The fractions were checked for lumazine synthaseactivity according to N). Enzymatic active fractions were collected andconcentrated using an ultra centrifuge (Beckman LE 70 with rotor 70Ti;32000 rpm, 18 h, 4° C.). The purity check was carried out according toExample 1 M) (SDS-PAGE) whereby just one band could be observed at amolecular weight of 16 kDa. The enzymatic activity was measuredaccording to Example 1 N), the protein concentration was determinedaccording to Example 1 O). Using these data a specific activity of 12400U/mg could be calculated. Negative staining shots according to Example 1P) showed hollow spherical particles with an outer diameter of 15 nm andan inner diameter of 5 nm.

[0271] T) To check the quarternary structure of the isolated purelumazine synthase experiments analogous to Example 1 S) to U) werecarried out yielding comparable results.

Example 3

[0272] Replacement of Cysteine 93 with Serine in the Lumazine Synthasefrom Bacillus subtilis Using Site Directed Mutagenesis

[0273] A) The gene coding for the lumazine synthase from Bacillussubtilis was amplified using the oligonucleotides PNCO-M2 (5′ aga tatttt cat taa aga gga gaa 3′) as forward primer, which is at his 3′-endidentical to ribosome binding site of the vector and which is deletingthe vector based EcoRI site at his 5′-end. As reverse primer theoligonucleotide RibH-3 (5′ tat tat gga tcc tta ttc aaa tga gcg gtt taaatt tg 3′) was used, which is at his 3′-end identical to the 3′-end ofthe ribH gene and which introduces a recognition site for theendonuclease BamHI (G*GATCC) directly after the stop codon. The plasmidpNCO-BS-LuSy (Example 1) was used as template for the PCR (Mullis etal., 1986).

[0274] 10 μl PCR-buffer (75 mM Tris/HCl, pH 9.0; 20 mM (NH₄)₂SO₄; 0.01%(w/v) Tween 20)

[0275] 6 μl Mg²⁺[1.5 mM]

[0276] 8 μl dNTP's [je 200 μM]

[0277] 1 μl PNCO-M2 [0.5 μM]

[0278] 1 μl RibH-3 [0.5 μM]

[0279] 1 μl pNCO-BS-LuSy [10 ng]

[0280] 1 μl Goldstar-Taq-Polymerase [0.5 U] (Eurogentec, Seraing,Belgien)

[0281] 72 μl H₂O_(bidest)

[0282] PCR cycle protocol (GeneAmp® PCR System 2400; Perkin Elmer):

[0283] 1. 5.0 min 95° C.

[0284] 2. 0.5 min 94° C.

[0285] 3. 0.5 min 50° C.

[0286] 4. 0.5 min 72° C.

[0287] 5. 7.0 min 72° C.

[0288] 6. ∞ 4° C.

[0289] Steps 2.-4. were repeated 20 times.

[0290] B) The PCR mixture was analysed and purified analogous to Example1 B) yielding a DNA fragment with a length of 505 bp.

[0291] C) A part of the ribH gene coding for the lumazine synthase fromBacillus subtilis was amplified using the oligonucleotides PNCO-M1(5′gtg agc gga taa caa ttt cac aca g 3′) as forward primer, which annealsto the vector sequence in 5′ direction of the EcoRI site at position 88,and C93S (5′ gca gct tca ttc gaa aca taa tcg taa tg 3′), which isresponsible for the replacement of the amino acid residue cysteine 93 byserine via site directed mutagenesis and which is introducing a new sitefor the restriction endo nuclease BstBI for the detection of themutation. The plasmid pNCO-BS-LuSy (Example 1) was used as template forthe PCR (Mullis et al., 1986). The PCR protocol, the analysis and thepurification of the PCR mixture was carried out analogous to A) and B)yielding a DNA fragment with a length of 256 bp.

[0292] D) Extension of the DNA-fragment (256 bp, containing the mutationand an intact EcoRI site at the 5′ end) from C) via combination with theDNA-fragment (505 bp, representing the total ribH, but with a deletedEcoRI site at the 5′ end) from B) and PCR. Equimolar amounts (each 500fmol) of the DNA fragments from B) and C) served as primers in the PCR.

[0293] 10 μl buffer

[0294] 6 μl Mg²⁺[1.5 mM]

[0295] 8 μl dNTP's [each 200 μM]

[0296] 1 μl DNA-fragment from B) (500 fmol)

[0297] 1 μl DNA-Fragment from C) (500 fmol)

[0298] 1 μl Goldstar-Taq-polymerase [0.5 U]

[0299] 73 μl H₂O_(bidest)

[0300] PCR cycle protocol (GeneAmp® PCR System 2400; Perkin Elmer):

[0301] 1. 5.0 min 95° C.

[0302] 2. 0.5 min 94° C.

[0303] 3. 0.5 min 65° C.

[0304] 4. 0.5 min 72° C.

[0305] 5. 7.0 min 72° C.

[0306] 6. ∞ 4° C.

[0307] Steps 2.-4. were repeated 20 times.

[0308] E) An aliquot of the PCR mixture from D) served as template for aPCR using the oligonucletides PNCO-M1/RibH-3 as forward and as reverseprimers. The PCR was carried out analogous A), excepting that the steps2.-4. were repeated 25 times.

[0309] F) The PCR mixture was analysed and purified analogous Example 1B), yielding a DNA-fragment with 528 bp.

[0310] G) The further handling was carried out analogous Example 1E)-I). The presence of the mutation was checked via digestion of theisolated plasmid pNCO-BS-LuSy-C93S with the restriction endonucleaseBstBI (TT*CGAA) yielding DNA fragments with 3698 bp and 181 bp.

[0311] H) The DNA sequencing was carried out analogous to Example 1 J).

[0312] I) The isolation of the protein and the quality checks werecarried out analogous to Example 1 K)-S) yielding compareable results,meaning that there were no significant differences to wild type lumazinesynthase.

Example 4

[0313] Replacement of Cysteine 139 with Serine in the Lumazine Synthasefrom Bacillus subtilis Using Site Directed Mutagenesis

[0314] A) The gene coding for the lumazine synthase from Bacillussubtilis was amplified analogous Example 3 A) using the oligonucleotidesPNCO-M2 and RibH-3 as primers and the plasmid pNCO-BS-LuSy (Example 1)as template and purified analogous Example 1 B) yielding a DNA fragmentwith 505 bp.

[0315] B) A part of the ribH gene coding for the lumazine synthase fromBacillus subtilis was amplified using the oligonucleotides PNCO-M1(5′gtg agc gga taa caa ttt cac aca g 3′) as forward primer, which annealsto the vector sequence in 5′ direction of the EcoRI site, and C139S (5′ggc aga aac agc tga atc tac acc ttt gtt g 3′), which is responsible forthe replacement of the amino acid residue cysteine 139 by serine viasite directed mutagenesis and which is introducing a new site for therestriction endo nuclease PvuII for the detection of the mutation. Theplasmid pNCO-BS-LuSy (Example 1) was used as template for the PCR(Mullis et al., 1986). The PCR protocol, the analysis and thepurification of the PCR mixture was carried out analogous to Example 3A) and Example 1 B) yielding a DNA fragment with a length of 394 bp.

[0316] C) The further handling was carried out analogous to Example 3 D)—H). The mutation was checked via digestion of the plasmidpNCO-BS-LuSy-C139S with the restriction endonuclease PvuII (CAG*CTG)yielding DNA fragments with 3539 bp and 340 bp.

[0317] D) The isolation of the protein and the quality checks werecarried out analogous to Example 1 K) to S) yielding compareableresults, meaning that there were no significant differences to wild typelumazine synthase.

Example 5

[0318] Replacement of Cysteine 93 and 139 with Serine in the LumazineSynthase from Bacillus subtilis Using Site Directed Mutagenesis

[0319] A) The construction of the double mutant plasmidpNCO-BS-LuSy-C93/139S was carried out analogous Example 4, exceptingthat the plasmid pNCO-BS-LuSy-C93S was used as template for the PCR.

[0320] B) The isolation of the protein and the quality checks werecarried out analogous to Example 1 K)-S) yielding compareable results,meaning that there were no significant differences to wild type lumazinesynthase.

[0321] Construction of Expression Vectors for the Fusion of Proteins tothe N- and to the C-Terminus of the Lumazine Synthase from Bacillussubtilis

[0322] The following examples describe the preparation of Escherichiacoli expression vectors for the fusion of genes or synthetic DNAfragments to the 5′- or the 3′-end of the ribH gene coding for thelumazine synthase from Bacillus subtilis. According to the invitationthe plasmid contains the following prefered vector elements: A promotorsequence from the bacteriophage T5, an operator sequence from thelac-operon from Escherichia coli, an ampicilline resistance marker geneand an Escherichia coli plasmid origin of replication.

Example 6

[0323] Vector for the Fusion of DNA Coding for a Target Peptide to the5′-End of the ribH Gene (Coding for the Lumazine Synthase) from Bacillussubtilis

[0324] A) The gene coding for the lumazine synthase from Bacillussubtilis was amplified analogous Example 1 A), excepting that theoligonucleotide N1 (5′ act atg gcg gcg gcg cgt agc tgc gcg gcc gct atgaat atc ata caa gga aat tta g 3′), which introduced a recognition sitefor the restriction endonuclease NotI (GC*GGCCGC) in close contact tothe start codon of the ribH gene, was used as forward primer and theoligonucleotide RibH-4 (3′ tat tat gga tcc aaa tta ttc aaa tga gcg gtttaa att tg 3′) which introduced a recognition site for the endonucleaseBamHI (G*GATCC) in close distance to the stop codon, was used as reverseprimer. The plasmid pRF2 (Example 1) was used as template for the PCR.

[0325] B) The PCR mixture was analyzed and purified analogous to Example1 B) yielding a DNA-fragment with 513 bp.

[0326] C) 10 ng of the isolated DNA fragment from B) served as atemplate for a second PCR using the oligonucleotide N2 (5′ ata ata gaattc att aaa gag gag aaa tta act atg gcg gcg gcg cgt agc tgc 3′), whichextended the DNA fragment from B) in 5′ direction whereby a ribosomebinding site and a recognition site for the restriction endonucleaseEcoRI (G*AATTC) was introduced as forward primer and the oligonucleotideRibH-4 as reverse primers.

[0327] D) The further handling was carried out analogous to Example 1B), E)-J) yielding the plasmid pNCO-N-BS-LuSy.

Example 7

[0328] Vector for the Fusion of DNA Coding for a Target Peptide to the3′-end of the ribH Gene (Coding for the Lumazine Synthase) from Bacillussubtilis

[0329] A) The gene coding for the lumazine synthase from Bacillussubtilis was amplified analogous to Example 1 A), excepting that theoligonucleotide EcoRI-RBS-2 (Example 2 A)) was used as forward primerand oligonucleotide C2 (5′ ttt tcg gga tcc ttt taa act gtt tgc ggc cgctaa ttc aaa tga gcg gtt taa att tg 3′), which introduced a new site forthe restriction endonuclease NotI in close contact to the last codingbase triplett of the ribH gene and which introduced a new recognitionsite for the restriction endonuclease BamHI in a distance of 13nucleotides downstream to the NotI site, was used as reverse primer andthe plasmid pNCO-BS-LuSy (Example 1) was used as template for the PCR.The stop codon of the wild type ribH gene was replaced by the basetriplett TTA coding for the amino acid residue leucine.

[0330] B) The further handling was carried out analogous to Example 1B), E)-J) yielding the plasmid pNCO-C-BS-LuSy.

[0331] Fusion of Complete ORFs (Open Reading Frames) to the N-Terminusor to the C-Terminus of the Lumazine Synthase from Bacillus subtilis

[0332] The following examples describe the fusion of complete genes tothe 5′- or the 3′-end of the ribH gene coding for the lumazine synthasefrom Bacillus subtilis. These examples illustrate the feasibility tofuse complete, biological active target proteins to the N-terminus or tothe C-terminus of the icosahedral lumazine synthase from Bacillussubtilis.

Example 8

[0333] Fusion of the Dihydrofolate Reductase (folA; DHFR) fromEscherichia coli to the N-Terminus of the Lumazine Synthase (ribH) fromBacillus subtilis

[0334] A) The gene coding for the dihydrofolate reductase (DHFR) fromEscherichia coli was amplified analogous to Example 1 A), excepting thatthe oligonucleotide EC-DHFR-1 (5′ gag gag aaa tta act atg atc agt ctgatt gcg g 3′), which bound at its 3′-end to the 5′-end of the folA geneand which introduced a part of an optimized ribosome binding siteupstream to the start codon, was used as forward primer and theoligonucleotide EC-DHFR-2 (5′ cta gcc gta aat tct ata gcg gcc gca cgccgc tcc aga atc 3′), which bound at its 3′-end to the 3′-end of the DHFRgene and which introduced a new recognition site for the restrictionendonuclease NotI directly after the last coding base triplett of thefolA gene, was used as reverse primer. Circa 50 ng of isolatedchromosomal Escherichia coli DNA (RR28) were used as template for thePCR.

[0335] B) The PCR mixture was analyzed and purified analogous to Example1 B) yielding a DNA-fragment with 513 bp.

[0336] C) A second PCR was carried out analogous to Example 1 C),excepting that the oligonucleotide BS-MfeI (5′ ata ata caa ttg att aaagag gag aaa tta act atg 3′), which extended the ribosome binding site in5′-direction and which introduced a site for the restrictionendonuclease MfeI (C*AATTG) was used as forward primer and theoligonucleotide EC-DHFR-2 was used as reverse primer.

[0337] D) The PCR mixture was analyzed and purified analogous to Example1 B) yielding a DNA-fragment with 531 bp.

[0338] E) The isolated DNA fragment from D) was digested using therestriction endonuclease MfeI (the DNA-overhang generated by Mfel(C*AATTG) is compatible with the DNA-overhang generated by EcoRI(G*AATTC)).

[0339] 30.0 μl DNA fragment from D)

[0340] 5.0 μl MfeI [50 U]

[0341] 10.0 μl buffer 4 (10×; 50 mM K-acetate, 20 mM tris-acetate, 10 mMMg-acetate, 1 mM dithiothreitol, pH 7.9) 55.0 μl H₂O_(bidest)

[0342] The enzymes were purchased from New England Biolabs (Schwalbach,Germany). The mixture was incubated for 150 min at 37° C. Afterincubation the mixture was purified as described under Example 1 B) andused for the digestion with the restriction endonuclease NotI.

[0343] F) In a second step the purified DNA fragment from E) wasdigested with the restriction endonuclease NotI.

[0344] 30.0 μl DNA fragment from E)

[0345] 5.0 μl NotI [50 U]

[0346] 10.0 μl buffer 3 (10×; 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl₂,1 mM dithiothreitol, pH 7.9)

[0347] 55.0 μl H₂O_(bidest)

[0348] The enzymes were purchased from New England Biolabs (Schwalbach,Germany). The mixture was incubated for 150 min at 37° C. Afterincubation the mixture was purified as described under Example 1 B) andused in a ligation protocol.

[0349] G) In a first step 5 μg of the expression vector pNCO-N-BS-LuSyin a volume of 30 μl were digested with the restriction endonucleaseNotI analogous to F) and purified analogous to Example 1 B).

[0350] H) In a second step the DNA fragment from G) was digested withthe restriction endonuclease EcoRI.

[0351] 30.0 μl vector-fragment from G)

[0352] 2.5 μl EcoRI [62,5 U]

[0353] 20.0 μl OPAU (10×; 500 mM K-acetate, 100 mM Mg-acetate, 100 mMTris-acetate, pH 7.5)

[0354] 47.5 μl H₂O_(bidest)

[0355] The enzymes were purchased from New England Biolabs (Schwalbach,Germany). The mixture was incubated for 150 min at 37° C. Afterincubation the mixture was purified as described under Example 1 B)yielding a fragment with a length of 3863 bp and used for in a ligationprotocol.

[0356] I) The further handling was carried out analogous to Example 1G)-L) yielding the plasmid pNCO-EC-DHFR-BS-LuSy.

[0357] J) The SDS-PAGE was carried out analogous to Example 1 M). In thecrude lysate of the strain XL1-pNCO-EC-DHFR-BS-LuSy an overexpressedprotein band with a molecular weight of circa 34.5 kDa could beobserved, which was not detectable in a strain without the plasmidpNCO-EC-DHFR-BS-LuSy. The expression rate of this protein could beestimated to 40-50% (concerning to the total soluble cell proteins).

[0358] K) The enzymatic activity was measured according to Example 1 N),the protein concentration was determined according to Example 1 O).Using these data a specific activity of circa 3700 U/mg could becalculated in the crude lysate which was campareable with recombinantwild type lumazine synthase.

[0359] L) The isolation of the fusion protein was carried out analogousto Example 2 S), excepting that the centrifugation of the protein in anultra centrifuge was carried out at 28000 rpm. Negative stainingexperiments were carried out analogous to Example 1 P), excepting thatthe pictures showed hollow spherical particles with an outer diameter ofcirca 20 nm and an inner diameter of circa 5 nm.

[0360] M) To check the quarternary structure of the isolated purelumazine synthase an experiment analogous to Example 1 S) was carriedout. It could be observed that the fusion protein (EC-DHFR-BS-LuSy),based on the increased diameter of the particle, migrates slower on thenative gel than the wild type lumazine synthase.

Example 9

[0361] Fusion of the Maltose Binding Protein (malE; MBP) fromEscherichia coli to the N-Terminus of the Lumazine Synthase (ribH) fromBacillus subtilis

[0362] A) The gene coding for the maltose binding protein fromEscherichia coli was amplified analogous Example 1 A), excepting thatthe oligonucleotide MALE-1 (5′ gag gag aaa tta act atg aaa atc gaa gaaggt aaa c 3′), which bound at its 3′-end to the 5′-end of the MBP geneand which introduced a part of an optimized ribosome binding siteupstream to the start codon, was used as forward primer andoligonucleotide MALE-2 (5′ gca ggt cga ctc tag cgg ccg cga att ctg 3′),which bound at its 3′-end to the 3′-end of the MBP gene and whichintroduced a new recognition site for the restriction endonuclease NotInearby the 5′-region of the MBP gene, was used as reverse primer. Circa10 ng of the plasmid pMAL-C2 (New England Biolabs, Schwalbach, Germany)were used as template for the PCR.

[0363] B) The PCR mixture was analyzed and purified analogous to Example1 B) yielding a DNA-fragment with 1210 bp.

[0364] C) A second PCR was carried out analogous to Example 1 C),excepting that the oligonucleotide BS-MfeI (5′ ata ata caa ttg att aaagag gag aaa tta act atg 3′), which extended the ribosome binding site in5′-direction and which introduced a recognition site for the restrictionendonuclease MfeI was used as forward primer and the oligonucleotideMALE-2 was used as reverse primer.

[0365] D) The PCR mixture was analyzed and purified analogous to Example1 B) yielding a DNA-fragment with 1227 bp.

[0366] E) The further handling was carried out analogous to Example 8E)-I) yielding the plasmid pNCO-EC-MBP-BS-LuSy.

[0367] F) The SDS-PAGE was carried out analogous to Example 1 M). In thecrude lysate of the strain XL1-pNCO-EC-MBP-BS-LuSy an overexpressedprotein band with a molecular weight of circa 59.5 kDa could beobserved, which was not detectable in a strain without the plasmidpNCO-EC-MBP-BS-LuSy. The expression rate of this protein could beestimated to 40-50% (related to the total soluble cell proteins).

[0368] G) The enzymatic activity was measured according to Example 1 N),the protein concentration was determined according to Example 1 O).Using these data a specific activity of circa 3700 U/mg could becalculated in the crude lysate which was comparable with recombinantwild type lumazine synthase.

[0369] H) The isolation of the fusion protein was carried out analogousto Example 2 S), excepting that the centrifugation of the protein in aultra centrifuge was carried out at 28000 rpm. Negative stainingexperiments were carried out analogous to Example 1 P), excepting thatthe pictures showed hollow spherical particles with an outer diameter ofcirca 25 nm and an inner diameter of circa 5 nm.

[0370] I) To check the quarternary structure of the isolated purelumazine synthase an experiment analogous to Example 1 S) was carriedout. It could be observed that the fusion protein (EC-MBP-BS-LuSy)—basedon the increased diameter of the particle—migrates slower on the nativegel than EC-DHFR-BS-LuSy or the wild type lumazine synthase.

Example 10

[0371] Fusion of the Dihydrofolate Reductase (folA, DHFR) fromEscherichia coli to the C-Terminus of the Lumazine Synthase (ribH) fromBacillus subtilis

[0372] A) The gene coding for the dihydrofolate reductase (DHFR) fromEscherichia coli was amplified analogous to Example 1 A), excepting thatthe oligonucleotide EC-FolA-1 (5′ ata gtg gcg aca atg cgg ccg ctg gtggag gcg gaa tga tca gtc tga ttg cgg cg 3′), which bound at its 3′-end tothe 5′-end of the DHFR gene and which introduced upstream to the startcodon of the DHFR gene a site for the restriction endonuclease NotI, wasused as forward primer and oligonucleotide EC-FolA-2 (5′ ttc tat gga tcctta ccg ceg ctc cag aat c 3′), which bound at its 3′-end to the 3′-endof the DHFR gene and which introduced a site for the restrictionendonuclease BamHI directly after the stop codon of the DHFR gene, wasused as reverse primer. Circa 50 ng of isolated chromosomal Escherichiacoli DNA (RR28) were used as template for the PCR.

[0373] B) The PCR mixture was analyzed and purified analogous to Example1 B) yielding a DNA-fragment with 524 bp.

[0374] C) The isolated DNA fragment from B) was digested using therestriction endonuclease BamHI.

[0375] 30.0 μl DNA fragment from B)

[0376] 3.0 μl BamHI [60 U]

[0377] 20.0 μl OPAU (10×)

[0378] 47.0 μl H₂O_(bidest)

[0379] The enzymes were purchased from Pharmacia Biotech (Freiburg,Germany). The mixture was incubated for 150 min at 37° C. After theincubation the mixture was purified as described under Example 1 B) andused for the digestion with the restriction endonuclease NotI.

[0380] D) In a second step the purified DNA fragment from C) wasdigested with the restriction endonuclease NotI analogous to Example 5F). After the incubation the mixture was purified as described underExample 1 B) and used for in a ligation protocol.

[0381] E) In a first step 5 μg of the expression vector pNCO-C-BS-LuSy(Example 4) in a volume of 30 μl were digested with the restrictionendonuclease NotI and BamHI analogous to C) and D). After the incubationthe mixture was purified as described under Example 1 B) yielding afragment with a length of 3880 bp and used for in a ligation protocol.

[0382] F) The further handling was carried out analogous to Example 1G)-L) yielding the plasmid pNCO-BS-LuSy-EC-DHFR.

[0383] G) The SDS-PAGE was carried out analogous to Example 1 M). In thecrude lysate of the strain XL1-pNCO-BS-LuSy-EC-DHFR an overexpressedprotein band with a molecular weight of circa 34.8 kDa could beobserved, which was not detectable in a strain without the plasmidpNCO-BS-LuSy-EC-DHFR. The expression rate of this protein could beestimated to 25% (related to the total soluble cell proteins).

[0384] H) The enzymatic activity was measured according to Example 1 N),the protein concentration was determined according to Example 1 O).Using these data a specific activity of circa 3700 U/mg could becalculated in the crude lysate which was campareable with recombinantwild type lumazine synthase.

[0385] I) The isolation of the fusion protein was carried out analogousto Example 2 S), excepting that the centrifugation of the protein in aultracentrifuge was carried out at 28000 rpm. Negative stainingexperiments were carried out analogous to Example 1 P), excepting thatthe pictures showed hollow spherical particles with an outer diameter ofcirca 20 nm and an inner diameter of circa 5 nm.

[0386] J) To check the quarternary structure of the isolated purelumazine synthase an experiment analogous to Example 1 S) was carriedout. It could be observed that the fusion protein(BS-LuSy-EC-DHFR)—based on the increased diameter of theparticle—migrated slower on the native gel than the wild type lumazinesynthase.

[0387] Linking of an Epitop (17 Aminoacid Residues) of the VP2 SurfaceProtein from a Mammal Virus to the N-Terminus, to the C-Terminus and toBoth Termini of the Lumazine Synthase from Bacillus subtilis

[0388] The following examples describe the fusion of short peptides toeather the N-terminus or the C-terminus or both termini of theicosahedral lumazine synthase from Bacillus subtilis under formation ofhollow spherical particles consisting of 60 subunits. Based on thefusion to both termini 120 epitops could be presented on the surface ofthe lumazine synthase.

[0389] The peptide with a length of 17 aa is a highly conserved part ofthe VP2 surface protein from different mammal viruses, e.g. ‘minkenteritis virus’, ‘feline panleukopenia virus’, ‘canine parvo virus’.

Example 11

[0390] Fusion of the VP2 Epitop to the N-Terminus of the LumazineSynthase (ribH) from Bacillus subtilis

[0391] A) The gene coding for the lumazine synthase from Bacillussubtilis was amplified analogous to Example 1 A), excepting thatoligonucleotide N-VP2-1 (5′ ggt cag ccg gct gtt cgt aac gaa cgt atg aatatc ata caa gga aat tta gtt ggt ac 3′), which bound at its 3′-end to the5′-end of the ribH gene and which coded for a part of the VP2 epitop atthe 5′-end, was used as forward primer and oligonucleotide RibH-3(Example 3) was used as reverse primer. The plasmid pRF2 served astemplate for the PCR.

[0392] B) The PCR mixture was analyzed and purified analogous to Example1 B) yielding a DNA-fragment with 504 bp and served as template for asecond PCR step.

[0393] C) 10 ng of the isolated DNA fragment from B) served as atemplate for a second PCR using the oligonucleotide N-VP2-2 (5′ gag gagaaa tta act atg ggg gac ggt gct gtt cag ccg gac ggt ggt cag ccg gct gttcgt aac gaa cg 3′), which extended the DNA coding for the VP2 epitopfrom B) in 5′ direction and which introduced a part of a ribosomebinding site, as forward primer and oligonucleotide RibH-3 as reverseprimer.

[0394] D) The PCR mixture was analyzed and purified analogous to Example1 B) yielding a DNA-fragment with 549 bp and served as template for athird PCR step.

[0395] E) The third PCR step was carried out analogous to Example 1 C),excepting that the oligonucleotides EcoRI-RBS-2 (Example 2 A) and RibH-3were used as forward and as reverse primers.

[0396] F) The PCR mixture was analyzed and purified analogous Example 1B), yielding a DNA-fragment with 567 bp.

[0397] G) The further handling was carried out analogous to Example 1E)-L) yielding the Escherichia coli expression strainXL1-pNCO-N-VP2-BS-LuSy.

[0398] H) The SDS-PAGE was carried out analogous to Example 1 M). In thecrude lysate of the strain XL1-pNCO-N-VP2-BS-LuSy an overexpressedprotein band with a molecular weight of circa 18.2 kDa could be observedwhich was not detectable in a strain without the plasmidpNCO-N-VP2-BS-LuSy. The expression rate of this protein could beestimated to 10% (related to the total soluble cell proteins).

[0399] I) The enzymatic activity was measured according to Example 1 N),the protein concentration was determined according to Example 1 O).Using these data a specific activity of circa 3700 U/mg could becalculated in the crude lysate which was campareable with recombinantwild type lumazine synthase.

Example 12

[0400] Fusion of the VP2 Epitop to the C-Terminus of the LumazineSynthase (ribH) from Bacillus subtilis

[0401] A) The gene coding for the lumazine synthase from Bacillussubtilis was amplified analogous to Example 1 A), excepting that theoligonucleotide C-VP2-1 (5‘cca ccg tcc ’ ggc tga aca gca ccg tca cct tcgaaa gaa cgg ttt aag ttt gcc 3′), which bound at its 3′-end to the 3′-endof the ribH gene and which introduced—directly after the last codingbase triplett of the ribH gene—a part of the DNA coding for the VP2epitop at its 5′-end, was used as reverse primer. The plasmid pRF2served as template for the PCR.

[0402] B) The PCR mixture was analyzed and purified analogous Example 1B), yielding a DNA-fragment with 506 bp, which served as template for asecond PCR step.

[0403] C) A second PCR was carried out analogous to Example 1 C),excepting that the oligonucleotide EcoRI-RBS-2 (Example 2 A)) was usedas forward primer and oligonucleotide C-VP2-2 (5′ ata tat gga tcc taacgt tcg tta cga aca gcc ggc tga cca ccg tcc ggc tga aca gca ccg tc 3′),which extended the DNA coding for the VP2 epitop in 3′-direction andwhich introduced a stop codon after the last coding base triplett of theVP2 epitop and a recognition site for the restriction endonuclease BamHI(G*GATCC), was used as reverse primer.

[0404] D) The PCR mixture was analyzed and purified analogous Example 1B), yielding a DNA-fragment with 564 bp.

[0405] E) The further handling was carried out analogous to Example 1E)-L) yielding the Escherichia coli expression strainXL1-pNCO-C-VP2-BS-LuSy.

[0406] F) The SDS-PAGE was carried out analogous to Example 1 M). In thecrude lysate of the strain XL1-pNCO-C-VP2-BS-LuSy an overexpressedprotein band with a molecular weight of circa 18.2 kDa could beobserved, which was not detectable in a strain without the plasmidpNCO-C-VP2-BS-LuSy. The expression rate of this protein could beestimated to 10% (related to the total soluble cell proteins).

[0407] G) The enzymatic activity was measured according to Example 1 N),the protein concentration was determined according to Example 1 O).Using these data a specific activity of circa 3700 U/mg could becalculated in the crude lysate which was comparable with recombinantwild type lumazine synthase.

Example 13

[0408] Fusion of the VP2 Epitop to the N-Terminus and to the C-Terminusof the Lumazine Synthase (ribH) from Bacillus subtilis

[0409] A) The expression plasmid pNCO-N-VP2-BS-LuSy (Example 11) servedas template for a PCR, which was carried out analogous to Example 12A)-D).

[0410] B) The further handling was carried out analogous to Example 1E)-L) yielding the Escherichia coli expression strainXL1-pNCO-N/C-VP2-BS-LuSy.

[0411] C) The SDS-PAGE was carried out analogous to Example 1 M). In thecrude lysate of the strain XL1-pNCO-N/C-VP2-BS-LuSy an overexpressedprotein band with a molecular weight of circa 20 kDa could be observed,which was not detectable in a strain without the plasmidpNCO-N/C-VP2-BS-LuSy. The expression rate of this protein could beestimated to 10% (related to the total soluble cell proteins).

[0412] D) The enzymatic activity was measured according to Example 1 N),the protein concentration was determined according to Example 1 O).Using these data a specific activity of circa 3700 U/mg could becalculated in the crude lysate which was comparable with recombinantwild type lumazine synthase.

Example 14

[0413] Linking of a Peptide (13 Aminoacid Residues; Bio-Peptide) to theC-Terminus of the Lumazine Synthase (ribH) from Bacillus subtilis

[0414] A) The gene coding for the lumazine synthase from Bacillussubtilis was amplified analogous to Example 1 A), excepting that theoligonucleotide EcoRI-RBS-2 (Example 2 A)) was used as forward primerand oligonucleotide C-Biotag-1 (5′ cat agc ttc gaa gat gcc gcc gag tgcggc cgc ttc gaa aga acg gtt taa gtt tgc cat ttc 3′), which bound at its3′-end to the 3′-end of the ribH gene and which introduced directlyafter the last coding base triplett of the ribH gene a DNA fragmentcoding for three alanines (linker residues) and which introduced in3′-direction to the DNA sequence coding for the linker residues a DNAfragment coding for a part of the Bio-Peptide, was used as reverseprimer. The plasmid pNCO-BS-LuSy (Example 1) served as template for thePCR.

[0415] B) The PCR mixture was analyzed and purified analogous Example 1B) yielding a DNA-fragment with 528 bp and served as template for asecond PCR step.

[0416] C) The second PCR step was carried out analogous to Example 1 C),excepting that the oligonucleotide EcoRI-RBS-2 (Example 2 A) was used asforward primer and oligonucleotide C-Biotag-2 (5′ tat tat gga tcc ttagcg cca ctc cat ctt cat agc ttc gaa gat gcc gcc gag tgc ggc 3′), whichextended the DNA sequence coding for the Bio-peptide in 3′-direction andwhich introduced a stop codon directly after this coding sequence andwhich introduced a recognition site for the restriction endonucleaseBamHI (G*GATCC), was used as reverse primer.

[0417] D) The PCR mixture was analyzed and purified analogous Example 1B) yielding a DNA-fragment with 558 bp.

[0418] E) The further handling was carried out analogous to Example 1E)-L) yielding the Escherichia coli expression strainXL1-pNCO-C-Biotag-BS-LuSy.

[0419] F) The enzymatic activity was measured according to Example 1 N),the protein concentration was determined according to Example 1 O) butno activity based on a recombinant expression of a lumazine synthasecould be detected.

[0420] G) The SDS-PAGE was carried out analogous to Example 1 M). In thecrude lysate of the strain XL1-pNCO-C-Biotag-BS-LuSy no overexpressedprotein band with a molecular weight of circa 18.5 kDa could beobserved.

[0421] H) To check the total expressed cell proteins (total cellextract, soluble an insoluble proteins) of the strainXL1-pNCO-C-Biotag-BS-LuSy cells were fermented and handled analogous toExample 1 K). 1/12 of the resulting cell pellet was suspended in 300 μlsample buffer (Example 1 M)) and incubated on a boiling water bath for15 min. After cooling down to 4° C. the suspension was centrifuged(15000 rpm, 5 min, 4° C.). 8 μl of the clear supernatant was applied toa SDS-PAGE analogous to Example 1 M). A recombinant protein band with amolecular weight of 18.5 kDa could be observed in the total cell extractof the strain XL1-pNCO-C-Biotag-BS-LuSy but in an insoluble form(inclusion bodies). The observed protein band corresponded to circa 15%of the total cell extract of the strain.

[0422] I) To verify that the observed recombinant protein bandcorresponds to the arteficial lumazine synthase fusion protein(C-Biotag-BS-LuSy) a western blot analysis was carried out analogous toExample 1 Q), excepting that in addition to the soluble cell extract thetotal cell extract was analyzed. After the development of thePVDF-membrane recombinant lumazine synthase fusion protein could bedetected mostly in the total cell but hardly in the soluble cellextracts.

[0423] J) Detection of the biotinylation of the fusion protein: Startingfrom a denaturating SDS-polyacryl amide gel (Example 1 M)) proteins weretransfered on a PVDF membrane by electro blotting (current: 40 mA, 2 h).After transferance of the proteins, the membrane was rinsed inantibody-washing-solution-A (Example 1 Q)). Afterwards the membrane wasincubated in antibody-washing-solution-B (Example 1 Q)) for 1 h at roomtemperature. Subsequent the membrane was incubated overnight in 15 mlantibody-washing-solution-C (Example 1 Q)) containing 20 μlstreptavidin-alkaline-phosphatase-conjugate (Promega, Madison, Wis.,USA). Afterwards the membrane was washed 3 times using each 5 mlantibody-washing-solution-A. The visualization of streptavidin bound tothe immobilized biotin was carried out using the substrates for thealkaline phosphatase. 50 μl BCIP-stock solution (25 mg5-bromo-4-chloro-3-indolyl phosphate (Sigma, Munich, Germany) solved in500 μl dimethylformamide, store at 4° C. in the dark) and 100 μlNBT-stock solution (50 mg nitro blue tetrazolium (Sigma, Munich,Germany) solved in a mixture of 700 μl dimethylformamide and 300 μlwater, store at 4° C. in the dark) were mixted together in 15 mlalkaline phosphatase buffer (100 mM tris-HCl, 100 mM NaCl, 5 mM MgCl₂,pH 9.5). The lumazine synthase with covalently bound biotin could bedetected on the membrane as a single blue band with a molecular weightof circa 18.5 kDa. This protein band couldn't be observed in anEscherichia coli strain without the expression plasmidpNCO-C-Biotag-BS-LuSy. The reaction of the alkaline phosphatase wasstopped via incubation of the membrane in 5 ml of stop solution (20 mMtris-HCl, 25 mM EDTA-Na₂, pH 8.0).

[0424] K) For the refolding of the expressed recombinant fusion proteinthe soluble protein fraction was removed analogous to example 1 L).

[0425] L) The pellet resulting from K) was incubated in refolding bufferA (100 mM K-phosphate, pH 7.0, 6 M urea, 6 mM5-nitro-6-(D-ribitylamino)2,4-(1H,3H)-pyrimidindione, 100 mMdithiothreitol (DTT)) for 24 h at room temperature. The resultingsolution was dialysed twice against the 10-fold volume of refoldingbuffer B (100 mM K-phosphate, pH 7.0, 1 mM5-nitro-6-(D-ribitylamino)2,4-(1H,3H)-pyrimidindione, 1 MM DTT) for 12 hat 4° C. The precipitated proteins in the dialysed solution were removedvia centrifugation (Sorvall SS34-rotor; 15000 rpm; 20 min; 4° C.). Thesoluble proteins in the resulting supernatant were concentrated using anultracentrifuge (Beckman TFT 70-rotor; 32000 rpm; 16 h; 4° C.). Theanalytic of the proteins was carried out analogous to J), Example 1 S)and Example 1 P) yielding an arteficial protein consisting of 60subunits forming an icosahedral structure.

[0426] M) To check the accessibility of the biotin molecules on thesurface of the icosahedron an ELISA protocol (enzyme linkedimmunosorbent assay) was carried out in microtiter plates 96 wells; 8wells in a column, 12 wells in a row). 100 μl avidin stock solution(Sigma, Munich, Germany) with a concentration of 1 mg/ml were diluted in20 ml coating buffer (20 mM Na-carbonate, pH 9.6) yielding the standardsolution. The wells of the microtiter plate were filled with 100 μlstandard solution and incubated overnight at room temperature.Subsequently the standard solution was removed and each well was washed3× with 200 μl PBS (20 mM Na-phosphate, 130 mM NaCl, pH 7.2). 350 μlSolution A (3% skimmed milk powder in PBS buffer) were added to eachwell and incubated for 1 h at 37° C. Afterwards Solution A was removedand 100 μl protein solution from L) (circa 1 mg/ml) was added to thefirst well of each column of the microtiter plate. 50 μl dilution buffer(1% skimmed milk powder in PBS) was added to the wells 2-8 in the samecolumn. In a subsequent step 50 μl of the protein solution in the firstwell was removed and added to the solution in well 2 and mixed with thedilution buffer. 50 μl of this diluted protein solution from well 2 wasremoved and added to the solution in well 3 and mixed. 50 μl from 3 to4, 50 μl from 4 to 5, 50 μl from 5 to 6, 50 μl from 6 to 7, 50 μl from 7to 8, 50 μl from 8 to waste (dilution: log 2). The samples wereincubated for 2 h at 37° C. Afterwards the solution in the wells wereremoved totally and the wells were washed 3× with 350 μl PBS.Subsequently 15 μl streptavidin-alkaline-phospatase conjugate (Promega,Madison, Wis., USA) were mixed with 20 ml dilution buffer (detectionsolution). To each well 100 μl Detection solution was added and themixture was incubated 1 h at 37° C. Afterwards the solution was removedtotally and the wells were washed 3× with 350 μl PBS. For thevisualization 150 μl substrate solution (10 mg p-nitrophenyl phosphate(Sigma, Munich, Germany) in 10 ml alkaline phosphatase buffer analogousto J)) were added to each well and incubated at room temperature. Theextinction was measured at 405 nm in an ELISA reader. The results showedbiotin molecules located on the surface of the lumazine synthase fusionprotein. The signal went through an optimum. If the concentration of thelumazine synthase fusion protein was highest a sterical hindrance forthe bindung of the streptavidin detection molecules could be observed.If the concentration was decreased, more and more biotin molecules gotaccessible for the streptavidin molecules and the measured signal gotmore intensive (going through an opimal concentration).

[0427] Labeling of of the C-Terminus of the Lumazine Synthase fromBacillus subtilis with a Reactive Amino Acid Residue

[0428] The following examples decribe the fusion of a reactive (forchemical reaction) amino acid residues (lysine and cysteine) to theC-terminus of the lumazine synthase from Bacillus subtilis. There aresome lysine residues located on the outer surface of the lumazine butthese residues are involved in structural elements of the capsid and notwell accessible for chemical reactions. There are no free cysteineresidues located on the outer surface of the lumazine synthase whichcould be used for chemical reaction via the thiole group.

[0429] To decrease the sterical hindrance and to increase theaccessibility of the reactive groups a linker (tentacle-linker; spacer)was introduced between the reactive amino acid and the C-terminus of thelumazine synthase.

Example 15

[0430] Extension of the C-Terminus of the Lumazine Synthase fromBacillus subtilis and Introduction of a Basic Amino Acid Residue(Lysine) as a Basis for the Chemical Coupling of Target Molecules

[0431] A) The gene coding for the lumazine synthase from Bacillussubtilis was amplified analogous to Example 1 A), excepting that theoligonucleotide EcoRI-RBS-2 (Example 2 A)) was used as forward primerand oligonucleotide C-Lys165 (5′ tat tat gga tcc tta ttt acc aga gcc accacc aga acc acc gcc acc ttc gaa aga acg gtt taa gtt tgc cat ttc 3′),which bound at its 3′-end to the 3′-end of the ribH gene and whichintroduced in close contact to the last coding base triplett of the ribHgene a DNA sequence coding for the peptide (Gly)₄Ser-(Gly)₃Ser-Gly-Lyswas used as reverse primer and the plasmid pNCO-BS-LuSy (Example 1) wasused as template for the PCR. Directly after the base triplett codingfor the lysine residue (aaa) at position 165 in the arteficial protein,a stop codon and a recognition site for the restriction endonucleaseBamHI was introduced.

[0432] B) The PCR mixture was analyzed and purified analogous to Example1 B) yielding a DNA-fragment with 543 bp.

[0433] C) The further handling was carried out analogous to Example 1B), E)-L) yielding the plasmid pNCO-Lys165-BS-LuSy.

[0434] D) The SDS-PAGE was carried out analogous to Example 1 M). In thecrude lysate of the strain XL1-pNCO-Lys165-BS-LuSy an overexpressedprotein band with a molecular weight of circa 17 kDa could be observedwhich was not detectable in a strain without the plasmidXL1-pNCO-Lys165-BS-LuSy. The expression rate of this protein could beestimated to 10% (related to all soluble cell proteins).

[0435] E) The further analytical experiments were carried out analogousto Example 1 N)-Q). No significant differences to the wild type lumazinesynthase could be observed with Lys165-BS-LuSy.

Example 16

[0436] Extension of the C-Terminus of the Lumazine Sythase from Bacillussubtilis and Introduction of a Amino Acid Residue with a SH-Group(Cysteine) as a Basis for the Chemical Coupling of Target Molecules

[0437] A) The construction was carried out analogous to Example 15A)-C), excepting that the oligonucleotide C-Cys167 (5 tat tat gga tcctta gca gcc acc acc aga gcc acc acc aga acc acc gcc acc ttc gaa aga acggtt taa gtt tgc cat ttc 3′), which bound at its 3′-end to the 3′-end ofthe ribH gene and which introduced in close contact to the last codingbase triplett of the ribH gene a DNA sequence coding for the peptide(Gly)₄Ser-(Gly)₃Ser-G1 _(Y3)-Cys was used as reverse primer and theplasmid pNCO-BS-LuSy (Example 1) was used as template for the PCR.Directly after the base triplett coding for the cysteine residue (tgc)at position 167 in the arteficial protein, a stop codon and arecognition site for the restriction endonuclease BamHI was introduced.

[0438] B) The PCR mixture was analyzed and purified analogous Example to1 B) yielding a DNA-fragment with 549 bp.

[0439] C) The further handling was carried out analogous to Example 1B), E)-L) yielding the plasmidpNCO-Cys167-BS-LuSy.

[0440] D) The SDS-PAGE was carried out analogous to Example 1 M). In thecrude lysate of the strain XL1-pNCO-Cys167-BS-LuSy an overexpressedprotein band with a molecular weight of circa 17.1 kDa could be observedwhich was not detectable in a strain without the plasmidXL1-pNCO-Cys167-BS-LuSy. The expression rate of this protein could beestimated to 5% (related to all soluble cell proteins).

[0441] E) The further characterization was carried out analogous toExample 1 N)-Q). No significant differences to the wild type lumazinesynthase could be observed with Cys167-BS-LuSy.

Example 17

[0442] Extension of the N-Terminus of the Lumazine Synthase fromBacillus subtilis via Introduction of a Peptide (12 Amino Acid Residues,FLAG-Tag) Serving as an Epitop for a Monoclonal Antibody(Anti-FLAG-M2/IgG1/Maus)

[0443] A) The gene coding for the lumazine synthase from Bacillussubtilis was amplified analogous Example 1 A), excepting that theoligonucleotide FLAG-BS-LuSy-1 (5′ ata ata ata aag ctt atg aat atc atacaa gga aat tta g 3′), which bound at its 3′-end to the 5′-end of theribH gene and which introduced a recognition site for the restrictionendonuclease HindIII (A*AGCTT) at the 5′-end, was used as forward primerand oligonucleotide Flag-BS-LuSy-2 (5′ tat tat gaa ttc tta ttc gaa agaacg gtt taa g 3′), which bound at its 3′-end to the 3′-end of the ribHgene and which introduced a recognition site for the restrictionendonuclease EcoRI (G*AATTC), was used as reverse primer. The plasmidpRF2 (Example 1 A)) served as template for the PCR.

[0444] B) The PCR mixture was analyzed and purified analogous Example 1B) yielding a DNA-fragment with 492 bp.

[0445] C) In a first step the isolated DNA fragment from B) and thevector pFLAG-MAC (Eastman Kodak Company, New Haven) were digested usingthe restriction endonuclease HindIII.

[0446] 30.0 μl DNA pFLAG-MAC [5 μg] resp. 30 μl DNA fragment from B)

[0447] 3.0 μl HindIII [60 U]

[0448] 10.0 μl OPAU (10×)

[0449] 57.0 μl H₂O_(bidest)

[0450] The enzymes was purchased from New England Biolabs (Schwalbach,Germany). The mixture was incubated for 150 min at 37° C. After theincubation the mixtures were purified as described under Example 1 B)and used for the digestion with the restriction endonuclease EcoRI.

[0451] D) In a second step the purified DNA fragments from C) weredigested with the restriction endonuclease EcoRI.

[0452] 30.0 μl DNA fragments from C)

[0453] 3.0 μl EcoRI [60 U]

[0454] 24.0 μl OPAU (10×)

[0455] 63.0 μl H₂O_(bidest)

[0456] The enzymes were purchased from New England Biolabs (Schwalbach,Germany). The mixture was incubated for 150 min at 37° C. After theincubation the mixture was purified as described under Example 1 B) andused in a ligation protocol.

[0457] E) The further handling was carried out analogous to Example 1G)-L) yielding the plasmid pFLAG-MAC-BS-LuSy.

[0458] F) The SDS-PAGE was carried out analogous to Example 1 M). In thecrude lysate of the strain XL1-pFLAG-MAC-BS-LuSy an overexpressedprotein band with a molecular weight of circa 17.7 kDa could beobserved, which was not detectable in a strain without the plasmidpFLAG-MAC-BS-LuSy. The expression rate of this protein could beestimated to 10% (related to all soluble cell proteins).

[0459] G) The enzymatic activity was measured according to Example 1 N),the protein concentration was determined according to Example 1 O).Using these data a specific activity of circa 3700 U/mg could becalculated in the crude lysate which was campareable with recombinantwild type lumazine synthase.

[0460] H) Negative staining experiments were carried out analogous toExample 1 P) yielding comparable results.

[0461] I) To check the binding properties of the fused FLAG-Peptide aWestern blot analysis analogous Example 1 Q) was carried out, exceptingthat the monoclonal antibody Anti-FLAG®M2′ (Eastman Kodak Company, NewHaven) was used as primary antibody (10 μl Anti-FLAG®M2 in 5 ml TBS (50mM Tris, 150 mM NaCl, pH 7.4)) and the monoclonal antibodyAnti-mouse-IgG-HRP-conjugate (10 μl Anti-mouse-IgG-HRP-conjugate (Sigma,Munich, Germany) in 5 ml TBS; see Example 18H)) was used as secondaryantibody. After visualization the fusion protein with a molecular weightof circa 17.7 kDa could be detected.

[0462] J) The purification of the fusion protein (FLAG-MAC-BS-LuSy) wascarried out analogous to Example 2 S), excepting that no lysozyme wasadded to the lysis buffer.

[0463] K) Negative staining experiments analogous to Example 1 P) showedhollow spherical particles with an outer diameter of circa 15 nm and aninner diameter of circa 5 nm.

[0464] L) The analysis of the quarternary structure was carried outanalogous to Example 1 S). The fusion protein migrated as a single bandwith minor changed mobility compared to wild type lumazine synthasebased on the slightly increased diameter.

Example 18

[0465] Linking of a Peptide (6 Histidin Residues; HIS6-Peptide), whichcan Serve as an Affinity Tag for the Binding to a Ni-Chelator AffinityMatrix or to a Monoclonal Antibody (Penta-His-Antibody) or to aNi-NTA-HRP-Conjugate, to the C-Terminus of the Lumazine Synthase fromBacillus subtilis

[0466] A) The gene coding for the lumazine synthase from Bacillussubtilis was amplified analogous to Example 1 A), excepting that theoligonucleotide RibH-His6-C-1 (5′ gtg gtg atg gtg atg ttc gaa aga acggtt taa g 3′), which bound at its 3′-end to the 3′-end of the ribH geneand which introduced directly after the last coding base triplett of theribH gene a DNA fragment coding for a part of the HIS6-Peptide, was usedas reverse primer. The plasmid pRF2 (see Example 1 A)) served astemplate for the PCR.

[0467] B) The PCR mixture was analyzed and purified analogous to Example1 B) yielding a DNA-fragment with 492 bp and served as template for asecond PCR step.

[0468] C) The second PCR step was carried out analogous to Example 1 C),excepting that the oligonucleotides EcoRI-RBS-2 (Example 2 A) was usedas forward primer and oligonucleotide RibH-His6-C-2 (5′ tat tat gga tcctta atg gtg gtg atg gtg atg 3′), which extended the DNA sequence codingfor the HIS6-peptide in 3′-direction and which introduced a stop codondirectly after this coding sequence and which introduced a recognitionsite for the restriction endonuclease BamHI (G*GATCC), was used asreverse primer.

[0469] D) The PCR mixture was analyzed and purified analogous Example 1B) yielding a DNA-fragment with 528 bp.

[0470] E) The further handling was carried out analogous to Example 1E)-L) yielding the Escherichia coli expression strainXL1-pNCO-C-His6-BS-LuSy.

[0471] F) The enzymatic activity was measured according to Example 1 N),the protein concentration was determined according to Example 1 O).Using these data a specific activity of circa 3700 U/mg could becalculated in the crude lysate which was campareable with recombinantwild type lumazine synthase.

[0472] G) To check the binding properties of the fused HIS6-Peptide aWestern blot analysis analogous Example 1 Q) was carried out, exceptingthat the monoclonal antibody ‘Penta-His™ Antibody’ (Qiagen, Hilden,Germany) was used as primary antibody (10 μl ‘Penta-His™ Antibody’ in 5ml TBS (Example 14 I)) and the monoclonal antibodyAnti-mouse-IgG-HRP-conjugate (10 μl Anti-mouse-IgG-HRP-conjugate in 5 mlTBS; Example 17 I)) was used as secondary antibody. After visualizationthe fusion protein could be detected at circa 17.1 kDa.

[0473] H) To check the accessibility of the HIS6-Peptide on the surfaceof the icosahedron an ELISA protocol (enzyme linked immunosorbent assay)was carried out on 96 well microtiter plates analogous to Example 14M)), excepting that the first well of the microtiter plate was filledwith 100 μl crude lysate from E) (5-8 mg/ml protein in the crudelysate). 50 μl Dilution buffer (1% skimmed milk powder in PBS) was addedto the wells 2-8 in the same column. In a subsequent step 50 μl of theprotein solution in the first well was removed and added to the solutionin well 2 and mixed with the dilution buffer. 50 μl of this dilutedprotein solution from well 2 was removed and added to the solution inwell 3 and mixed. 50 μl from 3 to 4, 50 μl from 4 to 5, 50 μl from 5 to6, 50 μl from 6 to 7, 50 μl from 7 to 8 and 50 μμl from 8 to waste(dilution: log 2). The samples were incubated overnight at 37° C.Afterwards the solution in the wells was removed totally and the wellswere washed 3× with 350 μl PBS. 350 μl Solution A (3% skimmed milkpowder in PBS buffer) were added to each well and incubated for 1 h at37° C. Subsequently Solution A was removed totally and each well waswashed 3× with 350 μl PBS. Afterwards 10 μl Penta-His™ Antibody (Qiagen,Hilden) were mixed with 5 ml Dilution buffer (1. Antibody solution). Toeach well 50 μl of the 1. Antibody solution were added and the mixturewas incubated 2 h at 37° C. Afterwards the solution was removed totallyand the wells were washed 3× with 350 μl PBS. In a further step 150 μlof the 2. Antibody solution (10 μl Anti-mouse-IgG-HRP-conjugate in 5 mlDilution buffer) were filled in each well and the mixture was incubated2 h at 37° C. Afterwards the solution was removed totally and the wellswere washed 3× with 350 μl PBS. For the visualization 150 μl Substratesolution (100 mg o-Phenylendiamine (Sigma, Munich, Germany) in 25 mlSubstrat buffer; Substrate buffer: 50 mM citric acid, pH 5) were addedto each well and incubated at room temperature. The extinction wasmeasured at 492 nm in an ELISA reader. The results showed that theHIS6-Peptides are located on the surface of the lumazine synthase fusionprotein. Based on the log 2 dilution of the target protein(C-His6-BS-LuSy), a decrease in the signal intensity could be observed.

[0474] I) Negative staining experiments analogous to Example 1 P) showedhollow spherical particles with an outer diameter of circa 15 nm and aninner diameter of circa 5 nm.

[0475] J) The analysis of the quarternary structure was carried outanalogous to Example 1 S). The fusion protein migrated as a single bandwith changed mobility compared to wild type lumazine synthase based onthe slight increase of the diameter.

Example 19

[0476] Preparation of a Mixed Lumazine Synthase Conjugate(Hetero-Oligomeric Lumazine Synthase Conjugates) Consisting of theLumazine Synthase Fusion Proteins C-Biotag-BS-LuSy and C-His6-BS-LuSyUsing an in Vitro Refolding Protocol

[0477] A) An Escherichia coli XL1 host strain carrying the expressionplasmid pNCO-C-Biotag-BS-LuSy (Example 14) was fermented analogous toExample 1 K, excepting that 500 ml medium was used.

[0478] B) An Escherichia coli XL1 host strain carrying the expressionplasmid pNCO-C-His6-BS-LuSy (Example 18) was fermented analogous toExample 1 K, excepting that 500 ml medium was used.

[0479] C) Cells from A) were thawed and lysed using a ultrasonic devicefrom Branson SONIC Power Company (Branson-Sonifier B-12A, Branson SONICPower Company, Dunbury, Conn.). The cell pellet from A) was suspended in40 ml Separation buffer (50 mM Tris pH 9.5) and cooled on ice for 10min. The cell suspension was then lysed using the ultrasonic device (15pulses at level 5). The suspension was then cooled on ice for 5 min andlysed under the same conditions again. The treatment was repeated 4times. After the last sonication the suspension was centrifuged(Sorvall-centrifuge with SS34-rotor; 5000 rpm, 4° C., 10 min), thesupernatant (crude lysate A-1) was removed and the cell pellet (cellpellet A-1) was used for the following steps.

[0480] D) The lysis was carried out for a second time analogous to C),whereas the cell pellet A-1 was suspended in 40 ml Separation buffer,yielding the crude lysate A-2 and the cell pellet A-2.

[0481] E) Cells from B) were thawed and lysed using a ultrasonic devicefrom Branson SONIC Power Company (Branson-Sonifier B-12A, Branson SONICPower Company, Dunbury, Conn.). The cell pellet from B) was suspended in40 ml Separation buffer (50 mM Tris pH 9.5) and incubated on ice for 10min. The cell suspension was then lysed using the ultrasonic device (15pulses at level 5). The suspension was then cooled on ice for 5 min andlysed under the same conditions again. The treatment was repeated 4times. After the last sonication the suspension was centrifuged(Sorvall-centrifuge with SS34-rotor; 15000 rpm, 4° C., 10 min) and thesupernatant (crude lysate B) was used for the following steps.

[0482] F) The cell pellet A-2 (C-Biotag-BS-LuSy) from D) was solubilizedin 40 ml Solubilization buffer (50 mM Tris, pH 9.5, 6 M guanidiniumthiocyanate (G-SCN), 100 mM dithiothreitole (DTT)) for 24 h at roomtemperature yielding the Solubilization solution.

[0483] G) To crude lysate B (40 ml) 6 M G-SCN and 100 mM DTE were addedand the mixture was incubated for 24 h at room temperature.

[0484] H) The Solubilization solution from F) was centrifuged(Sorvall-centrifuge with SS34-rotor; 15000 rpm, 25° C., 20 min) yieldingthe supernatant A-3 and the cell pellet A-3.

[0485] I) Afterwards aliquots of supernatant A-3 and cell pellet A-3were analyzed using a SDS-PAGE.

[0486] J) The supernatant was checked analogous Example 1 M).

[0487] K) For the analysis of cell pellet A-3 a small aliquot of cellpellet A-3 was suspended in 200 μl Sample buffer (Example 1 M) andboiled for 15 min. Afterwards the suspension was centrifuged (Eppendorffcentrifuge, 15000 rpm, 5 min, 4° C.) and 6 μl of the clear supernatantwas applied to a SDS-PAGE. The further handling was carried outanalogous to Example 1 M).

[0488] L) The data from J) and K) showed that the insoluble proteinC-Biotag-BS-LuSy could be solubilized to 80% under the describedconditions.

[0489] M) The cell pellet A-3 from H) was treated again following thesteps F) and H)-K). The amount of soluble material couldn't beincreased.

[0490] N) The concentrations of the fusion proteins from E) (crudelysate B) and H) (supernatant A-3) were in the same range (related tothe amount of target protein).

[0491] O) 2 ml of supernatant-C-Bio-BS-LuSy (supernatant A-3) and 2 mlof supernatant-C-His6-BS-LuSy (crude lysate B) were mixed (Mixture A).

[0492] P) 3.5 ml of supernatant-C-Bio-BS-LuSy (supernatant A-3) and 0.5ml of supernatant-C-His6-BS-LuSy (crude lysate B) were mixed (MixtureB).

[0493] Q) Mixture A and Mixture B were stirred for 48 h at roomtemperature.

[0494] R) Mixture A and Mixture B from Q) were dialysed against 400 mlSeparation buffer containing 8 M urea and 1 mM DTE for 18 h at roomtemperature.

[0495] S) 6.6 mM 5-Nitro-6-(D-ribitylamino)2,4-(1H,3H)-pyrimidindionewere added to Mixture A and Mixture B from S) yielding Mixture-A-Nitroand Mixture-B-Nitro. The solutions were stirred for 8 h at roomtemperature.

[0496] T) Mixture-A-Nitro and Mixture-B-Nitro were dialysed against 32ml (5×volume) Refolding buffer A (100 mM K-phosphate buffer, pH 7.0, 1mM 5-Nitro-6-(D-ribitylamino)2,4-(1H,3H)-pyrimidindione, 1 mM DTE, 0.02%Na-azide) for 12 h at 4° C. yielding Mixture-A-1/5 and Mixture-B-1/5.

[0497] U) In a subsequent step the Refolding buffer A from T) wasdiluted with 40 ml Refolding buffer B (100 mM K-phosphate buffer, pH7.0, 1 mM DTE, 0.02% Na-azide) and the dialysis was carried out forfurther 24 h at 4° C. yielding Mixture-A-1/10 and Mixture-B-1/10.

[0498] V) In a subsequent step the Refolding buffer B from U) wasdiluted with 80 ml Refolding buffer B and the dialysis was carried outfor further 24 h at 4° C. yielding Mixture-A-1/20 and Mixture-B-1/20.

[0499] W) Afterwards Mixture-A-1/20 and Mixture-B-1/20 were dialysed for24 h at 4° C. against 72 ml (10 fold volume) Refolding buffer C (100 mMK-phosphate buffer, pH 7.0, 0.25 mM5-Nitro-6-(D-ribitylamino)2,4-(1H,3H)-pyrimidindione, 1 mM DTE, 0.02%Na-azide) yielding Mixture-A-1/200 and Mixture-B-1/200.

[0500] X) Mixture-A-1/200 and Mixture-B-1/200 were centrifuged(Sorvall-centrifuge with SS34-rotor; 15000 rpm, 4° C., 20 min).

[0501] Y) Aliquots of the supernatants were analyzed using a SDS-PAGE.The analysis of the supernatants was carried out analogous to Example 1M).

[0502] Z) The analysis of the pellets resulting from X) was carried outanalogous to K).

[0503] AA) The analysis from Y) and Z) showed that mixed lumazinesynthase conjugates could be generated in an amount of 50-80% by thedescribed protocol. The amounts of the used protein concentrations atthe beginning of the refolding process corresponded to the amounts ofeach protein in the analyzed conjugates. No difference in the refoldingbehaviour of both proteins could be observed.

[0504] BB) To check the quarternary structure of the lumazine synthaseconjugates an experiment analogous to Example 1 S) was carried out,excepting that 40 μl protein solution (Supernatant Mixture-A-1/200;Supernatant Mixture-B-1/200) were used for the electrophoresis. Therefolded protein conjugates could be observed as single bands on thenative polyacrylamide gel. The mobility of both proteins was comparablewith the mobilities of the proteins C-Bio-BS-LuSy and C-His6-BS-LuSybased on the similar hydrodynamic diameter of the four proteins. Usingthe described protocol above refolded mixed lumazine synthase conjugatescontaining different fusion partners could be obtained.

[0505] CC) To check the presence of both fusion partners on the surfaceof a discret lumazine synthase conjugate an ELISA protocol (enzymelinked immunosorbent assay) was carried out on 96 wells microtiterplates. Coating of the micro titer wells with avidin was carried outanalogous to Example 14 M). After the coating process 100 μl (circa 0.5to 1 mg/ml) of the refolded protein from X) were filled in the 1. wellof a column. 50 μl Dilution buffer was added to the wells 2-8 in thesame column. In a subsequent step 50 μl of the protein solution in thefirst well was removed and added to the solution in well 2 and mixedwith the dilution buffer. 50 μl of this diluted protein solution fromwell 2 was removed and added to the solution in well 3 and mixed. 50 μlfrom 3 to 4, 50 μl from 4 to 5, 50 μl from 5 to 6, 50 μl from 6 to 7, 50μl from 7 to 8 and 50 μl from 8 to waste (dilution: log 2). The sampleswere incubated overnight at 37° C. Afterwards the solution in the wellswere removed totally and the wells were washed 3× with 350 μl PBS. 350μl Solution A (3% skimmed milk powder in PBS buffer) were added to eachwell and incubated for 1 h at 37° C. Subsequently the Solution A wasremoved totally and each well was washed 3× with 350 μl PBS. Afterwards10 μl Penta-His™ Antibody (Quiagen, Hilden) were mixed with 5 mlDilution buffer (1. Antibody solution). To each well 50 μl of the 1.Antibody solution were added and the mixture was incubated 2 h at 37° C.Afterwards the solution was removed totally and the wells were washed 3×with 350 μl PBS. In a further step 150 μl of the 2. Antibody solution(10 μl Anti-mouse-IgG-HRP-conjugate in 5 ml Dilution buffer) were filledin each well and the mixture was incubated 2 h at 37° C. Afterwards thesolution was removed totally and the wells were washed 3× with 350 μlPBS. For the visualization 150 μl Substrate solution (100 mgo-Phenylendiamin in 25 ml Substrat buffer; Substrate buffer: 50 mMcitric acid, pH 5) were added to each well and incubated at roomtemperature. The extention was measured at 492 nm in an ELISA reader.The data showed that the hetero-oligomeric lumazine synthase conjugatescould be bound to the avidin via biotin molecules on the surface of theicosahedron. On the other hand HIS6-Peptides could be detected via thehighly specific Penta-His-Antibody on the protein conjugates which werebound to the avidin coated microtiter plate via biotin. Based on the log2 dilution of the target protein (C-His6-BS-LuSy), a decrease in thesignal intensity could be observed. Supernatant mixture A-1/200(estimated: 30 HIS6-Peptides) showed a more intensive signal thanSupernatant mixture B-1/200 (estimated: 15 HIS6-Peptides). For thebinding of the hetero-oligomeric lumazine synthase conjugate to theavidin coated microtiter plate, just one single biotin molecule wasneeded. In the Supernatant mixture A-1/200 more HIS6-Peptides (30) werepresented on the surface of the icosahedron than in the Supernatantmixture B-1/200 (15). Based on this fact the signal resulting from thebinding of the Penta-His-Antibody should be more intensive in theSupernatant mixture A-1/200.

[0506] Using the described protocol above refolded mixed lumazinesynthase conjugates (heterooligomeric lumazine synthase conjugates)containing different fusion partners, whereby the fusion peptides arelocated on the surface of the icohedron, could be obtained.

Example 20

[0507] Construction of a Synthetic Gene Coding for a ThermostableLumazine Synthase Based on the Hyperthermophilic Bacterium Aquifexaeolicus (Deckert et al., 1998) 11 Oligonucleotides Adapted to theEscherichia coli Codon Usage for Highly Expressed Proteins Served asPrimers in a 6-Phase PCR

[0508] A) The gene coding for the lumazine synthase from Aquifexaeolicus was amplified using the oligonucleotide AQUI-1 (5′ gct gcg ggtgaa ctg gcg cgt aaa gag gac att gat gct gtt atc gca att ggc gtt ctc atc3′) as forward primer and oligonucleotide AQUI-2 (5′ cta atg aaa ggt tcgcga ggc ctt ttg aaa ctt cag agg cga tat aat cga aat gtg gcg ttg 3′) asreverse primer. Each of the oligonucleotides has been adapted to theEscherichia coli codon usage for highly expressed proteins (Grosjean undFiers, 1982; Ikemura, 1981; Wada et al. 1992). The oligonucleotideAQUI-1 contained a recognition site for the restriction endonucleaseMfeI (C*AATTG) and the oligonucleotide AQUI-2 contained a recognitionsite for the restriction endonuclease StuI (AGG*CCT). The plasmidpNCO-BS-LuSy (Example 1) served as template for the PCR.

[0509] 10 μl PCR-buffer (75 mM Tris/HCl, pH 9.0; 20 mM (NH₄)₂SO₄; 0.01%(w/v) Tween 20)

[0510] 6 μl Mg²⁺[1.5 mM]

[0511] 8 μl dNTP's [je 200 μM]

[0512] 1 μl AQUI-1 [0.5 μM]

[0513] 1 μl AQUI-2 [0.5 μM]

[0514] 1 μl pNCO-BS-LuSy [10 ng]

[0515] 1 μl Goldstar-Taq-Polymerase [0.5 U] (Eurogentec, Seraing,Belgien)

[0516] 72 μl H₂O_(bidest)

[0517] PCR cycle protocol (GeneAmp® PCR System 2400; Perkin Elmer):

[0518] 1. 5.0 min 95° C.

[0519] 2. 0.5 min 94° C.

[0520] 3. 0.5 min 50° C.

[0521] 4. 0.5 min 72° C.

[0522] 5. 7.0 min 72° C.

[0523] 6. ∞ 4° C.

[0524] Steps 2.-4. were repeated 20 times.

[0525] B) The PCR mixture was analysed and purified analogous Example 1B), excepting that an agarose gel was used containing 3% agarose,yielding a DNA-fragment with a length of 132 bp.

[0526] C) 10 ng of the purified DNA from B) served as a template for a2. PCR using the oligonucleotide AQUI-3 (5′ act ctg gtt cgt gtt cca ggctca tgg gaa ata ccg gtt gct gcg ggt gaa ctg gcg cgt aaa g 3′), which wasidentical to the 5′-end of primer AQUI-1 and the oligonucleotide AQUI-4(5′ cca agg tgt cag ctg taa taa cac cga agg tga tag gtt tac gta gtt ctaatg aaa ggt tcg cga ggc c 3′), which was identical to the 5′-end ofprimer AQUI-2, as forward and as reverse primers. The oligonucleotideAQUI-3 contained a recognition site for the restriction endonucleaseAgeI (A*CCGGT) and the oligonucleotide AQUI-4 a recognition site for therestriction endonucleases SnaBI (TAC*GTA) and PvuII (CAG*CTG). The PCRwas carried out analogous to A).

[0527] D) The PCR mixture was analysed and purified analogous B)yielding a DNA-fragment with a length of 219 bp.

[0528] E) 10 ng of the purified DNA from D) served as a template for a3. PCR using the oligonucleotide AQUI-5 (5′ gga ggg tgc aat tga ttg catagt ccg tca tgg cgg ccg tga aga aga cat tac tct ggt tcg tgt tcc agg c3′), which was identical to the 5-end of primer AQUI-3 and theoligonucleotide AQUI-6 (5′ gtt gcc gtg ttt tgt gcc ggc gcg ctc gat agcctg ttc caa ggt gtc agc tgt aat aac 3′), which was identical to the5′-end of primer AQUI-4, as forward and as reverse primers. Theoligonucleotide AQUI-5 contained a recognition site for the restrictionendonuclease EagI (C*GGCCG) and the oligonucleotide AQUI-6 a recognitionsite for the restriction endonucleases BssHII (G*CGCGC) and PvuII(CAG*CTG). The PCR was carried out analogous to A).

[0529] F) The PCR mixture was analysed and purified analogous B)yielding a DNA-fragment with a length of 309 bp.

[0530] G) 10 ng of the purified DNA from F) served as a template for a4. PCR using the oligonucleotide AQUI-7 (5′ cgg tat cgt agc atc acg ttttaa tca tgc tct tgt cga ccg tct ggt gga ggg tgc aat tga ttg cat ag 3′),which was identical to the 5′-end of primer AQUI-5 and theoligonucleotide AQUI-8 (5′ gaa taa gtt tgc cat ttc aat ggc aga aag cgctgc ttc cca acc ttt gtt gcc gtg ttt tgt gcc ggc 3′), which was identicalto the 5′-end of primer AQUI-6, as forward and as reverse primers. Theoligonucleotide AQUI-7 contained a recognition site for the restrictionendonuclease SalI (G*TCGAC) and the oligonucleotide AQUI-8 a recognitionsite for the restriction endonuclease Eco56I (G*CCGGC). The PCR wascarried out analogous to A).

[0531] H) The PCR mixture was analysed and purified analogous B)yielding a DNA-fragment with a length of 405 bp.

[0532] I) 10 ng of the purified DNA from H) served as a template for a5. PCR using the oligonucleotide AQUI-9 (5′ atg caa atc tac gaa ggt aaacta act gct gaa ggc ctt cgt ttc ggt atc gta gca tca cgt ttt aat c 3′),which was identical to the 5′-end of primer AQUI-7 and theoligonucleotide AQUI-10 (5′ tat tat gga tcc tta tcg gag aga ctt gaa taagtt tgc cat ttc aat gg 3′), which was identical to the 5′-end of primerAQUI-8, as forward and as reverse primers. The oligonucleotide AQUI-9contained a recognition site for the restriction endonuclease StuI(AGG*CCT) and the oligonucleotide AQUI-10 introduced a recognition sitefor the restriction endonuclease BamHI (G*GATCC) directly after the stopcodon of the gene coding for the lumazine synthase from Aquifexaeolicus. The PCR was carried out analogous to A).

[0533] J) The PCR mixture was analysed and purified analogous B)yielding a DNA-fragment with a length of 476 bp.

[0534] K) 10 ng of the purified DNA from J) served as a template for a6. PCR using the oligonucleotide AQUI-11 (5′ ata ata gaa ttc att aaa gaggag aaa tta act atg caa atc tac gaa ggt aaa cta ac 3′), which wasidentical to the 5′-end of primer AQUI-9 and which coded for anoptimized ribosome binding site at its 5′-end and the oligonucleotideAQUI-10, as forward and as reverse primers. The oligonucleotide AQUI-11contained a recognition site for the restriction endonuclease EcoRI(G*AATTC) upstream to the ribosome binding site. The PCR was carried outanalogous to A).

[0535] L) The PCR mixture was analysed and purified analogous B)yielding a DNA-fragment with a length of 510 bp.

[0536] M) The further handling was carried out analogous to Example 1 E)to G) yielding the plasmid pNCO-AA-LuSy.

[0537] N) The further handling of the plasmid pNCO-AA-LuSy was carriedout analogous to Example 1H) to M). In the crude lysate of the strainXL1-pNCO-AA-LuSy a protein band with a molecular weight of circa 16.7kDa could be observed. This protein band couldn't be observed in aEscherichia coli strain without the expression plasmid pNCO-AA-LuSy. Theobserved protein band corresponded to circa 20% of the total solubleproteins of the Escherichia coli strain.

[0538] O) The enzymatic activity was measured according to Example 1 N),whereby the protein showed an activity optimum in a temperature range of80-90° C.

[0539] P) Negative staining experiments were carried out analogous toExample 1 P) yielding spherical hollow protein particles with an outerdiameter of circa 15 nm and an inner diameter of circa 5 nm.

[0540] Q) The isolation of the lumazine synthase coded by the describedsynthetic gene was carried out in two steps: The fermentation of theEscherichia coli strain XL1-pNCO-AA-LuSy was carried out analogous toExample 1 K), excepting that 1 l medium was used. The lysis of theresulting cells was carried out analogous to Example 1 R). Thesupernatant after the centrifugation (crude lysate) was treated at 90°C. for 20 min in a water bath. Subsequently the resulting suspension wascentrifuged (Sorvall SS34-rotor; 15000 rpm; 4° C.; 30 min) and thesupernatant was used for the further experiments. The resultingrecombinant protein was 80% pure after this step. The furtherpurification was carried out analogous to Example 2 S) using agelfiltration column.

[0541] R) The quarternary check was carried out analogous to Example 1S) yielding a result comparable to the lumazine synthase capsids fromBacillus subtilis.

Example 21

[0542] Linking of a Peptide (13 Aminoacid Residues; Bio-Peptide), whichcan be Biotinylated in Vivo to the C-Terminus of the Lumazine Synthase(ribH) from Aquifex aeolicus (see Example 20 and 14)

[0543] A) The gene coding for the lumazine synthase from Aquifexaeolicus was amplified analogous to Example 1 A), excepting that theoligonucleotide EcoRI-RBS-2 (Example 2 A)) was used as forward primerand oligonucleotide AQUI-C-NotI (5′ tat tat tat agc ggc cgc tcg gag agactt gaa taa g 3′) was used as reverse primer. The oligonucleotideAQUI-C-NotI was at its 3′-end identical to the 3′-end of the ribH geneand introduced a recognition site for the restriction endonuclease NotI(GC*GGCCGC) directly after the last coding base triplett. The DNAsequence representing the recognition site for the endonuclease wastranslated into three alanine residues. The plasmid pNCO-AA-LuSy(Example 20) served as template for the PCR.

[0544] B) The PCR mixture was analyzed and purified analogous to Example1 B) yielding a DNA-fragment with 513 bp.

[0545] C) The DNA fragment from B) was digested with the restrictionendonuclease NotI analogous to Example 8 F). After incubation the DNAfragment was purified analogous Example 1 B).

[0546] D) The DNA fragment from C) was digested with the restrictionendonuclease EcoRI analogous to Example 5H). After incubation the DNAfragment was purified analogous Example 1 B) yielding a DNA fragmentwith a length of 498 bp.

[0547] E) 5 μg of the expression plasmid pNCO-C-Biotag-BS-LuSy (Example14), in a volume of 30 μl were treated analogous to Example 8 G) and H).The DNA fragment with a length of 3437 bp was isolated analogous toExample 1 B).

[0548] F) The further handling was carried out analogous to Example 1 G)to L) yielding the Escherichia coli expression strainXL1-pNCO-C-Biotag-AA-LuSy.

[0549] G) The SDS-PAGE was carried out analogous to Example 1 M). In thecrude lysate of the strain XL1-pNCO-C-Biotag-AA-LuSy no overexpressedprotein band with a molecular weigth of circa 18.5 kDa could beobserved.

[0550] H) The further handling was carried out analogous to Example 14H)to M) yielding comparable results.

Example 22

[0551] Linking of a Peptide (13 Aminoacid Residues; Bio-Peptide), whichcan be Biotinylated in Vivo, via a Linker Peptide Consisting of theAminoacid Residues H-H-H-H-H-H-A-A-A to the C-Terminus of theThermostable Lumazine Synthase (ribH) from Aquifex aeolicus

[0552] A) The gene coding for the lumazine synthase from Aquifexaeolicus was amplified analogous to Example 1 A), excepting that theoligonucleotide EcoRI-RBS-2 (Example 2 A)) was used as forward primerand oligonucleotide AQUI-C-HIS₆-NotI (5′ tat tat tat agc ggc cgc atg gtggtg atg gtg atg tcg gag aga ctt gaa taa gtt tgc 3′) was used as reverseprimer. The oligonucleotide AQUI-C-HIS₆-NotI was at its 3′-end identicalto the 3′-end of the ribH gene and introduced directly after the lastcoding base triplett of the ribH gene a sequence coding for 6 histidineresidues and directly after this sequence a recognition site for therestriction endonuclease NotI (GC*GGCCGC). The DNA sequence representingthe recognition site for the endonuclease was translated into threealanine residues. The plasmid pNCO-AA-LuSy (Example 20) served astemplate for the PCR.

[0553] B) The PCR mixture was analyzed and purified analogous to Example1 B) yielding a DNA-fragment with 531 bp.

[0554] C) The DNA fragment from B) was digested with the restrictionendonuclease NotI analogous to Example 8 F). After incubation the DNAfragment was purified analogous Example 1 B).

[0555] D) The DNA fragment from C) was digested with the restrictionendonuclease EcoRI analogous to Example 8 H). After incubation the DNAfragment was purified analogous Example 1 B) yielding a DNA fragmentwith a length of 516 bp.

[0556] E) The expression vector was treated analogous to Example 21 E).

[0557] F) The further handling was carried out analogous to Example 1 G)to J) yielding the Escherichia coli expression strainXL1-pNCO-HIS6-C-Biotag-AA-LuSy.

[0558] G) The fermentation of the cells was carried out analogous toExample 1 R). After the centrifugation the clear supernantant wasremoved and the resulting cell pellet was used for the furtherexperiments.

[0559] H) The insoluble pellet from G) was incubated in 50 mlNTA-buffer-A (50 mM Na-phosphate-buffer pH 8.0, 300 mM NaCl, 0.02%Na-azide, 6 M guanidiniumhydrochloride) for 24 h at room temperaturewhereby the solution was stirred. Afterwards the suspension wascentrifuged (Sorvall SS34-Rotor, 15000 rpm, 20° C., 20 min). Theresulting supernatant was removed and used for the further experiments.The supernatant was mixed with 6 ml Ni-NTA-agarose (Qiagen, Hilden,Germany) and incubated overnight in a waver (20° C.). Afterwards thesuspension was centrifuged (800 g, 20° C., 10 min). The supernatant wasremoved and the resulting pellet was suspended in 10 ml NTA-buffer-A andincubated for 15 min in a waver (20° C.). The suspension was centrifugedagain and the supernatant was removed. The pellet was suspended in 10 mlNTA-buffer-B (8 M urea, 100 mM Na-phosphat-buffer, 10 mM Tris pH 6.3)and incubated for 15 min in a waver (20° C.) and subsequentlycentrifuged. The treatment was repeated once more. Subsequently theresulting pellet was washed twice using each 10 ml NTA-buffer-C (8 Murea, 100 mM Na-phosphat-Puffer, 10 mM Tris pH 5.9). At least the pelletwas treated twice using each 10 ml NTA-Puffer-D (8 M urea, 100 mMNa-phosphat-buffer, 10 mM Tris pH 4.5). The pollutions could be removedusing NTA-buffer-B and the target protein could be eluted by the use ofNTA-buffer-C and NTA-buffer-D. After neutralization of the fractions aSDS-PAGE was carried out. On the polyacrylamide gel just one single bandwith a molecular weight of 19.3 kDa could be observed.

Example 23

[0560] Linking of a Peptide (13 Aminoacid Residues; Bio-Peptide), whichcan be Biotinylated in Vivo, via a Linker Peptide Consisting of theAminoacid Residues H-H-H-H-H-H-G-G-S-G-A-A-A to the C-Terminus of theThermostable Lumazine Synthase (ribH) from Aquifex aeolicus

[0561] A) The gene coding for the lumazine synthase from Aquifexaeolicus was amplified analogous to Example 21), excepting that theoligonucleotide EcoRI-RBS-2 (Example 2 A)) was used as forward primerand oligonucleotide AQUI-C-HIS₆-GLY₂)-SER-GLY-NotI (5′ tat tat tat agcggc cgc gcc aga acc gcc atg gtg gtg atg gtg atg tcg gag aga ctt gaa taagtt tgc 3′) was used as reverse primer. The oligonucleotideAQUI-C-HIS₆-GLY₂-SER-GLY-NotI was at its 3′-end identical to the 3′-endof the ribH gene and introduced directly after the last coding basetriplett of the ribH gene a sequence coding for the peptideH-H-H-H-H-H-G-G-S-G and directly after this sequence a recognition sitefor the restriction endonuclease NotI (GC*GGCCGC). The DNA sequencerepresenting the recognition site for the endonuclease was translatedinto three alanine residues. The plasmid pNCO-AA-LuSy (Example 20)served as template for the PCR. The PCR was carried out analogous toExample 1 A).

[0562] B) The PCR mixture was analyzed and purified analogous to Example1 B) yielding a DNA-fragment with 543 bp.

[0563] C) The DNA fragment from B) was digested with the restrictionendonuclease NotI analogous to Example 8 F). After incubation the DNAfragment was purified analogous Example 1 B).

[0564] D) The DNA fragment from C) was digested with the restrictionendonuclease EcoRI analogous to Example 8H). After incubation the DNAfragment was purified analogous Example 1 B) yielding a DNA fragmentwith a length of 528 bp.

[0565] E) The expression vector was treated analogous to Example 21 E).

[0566] F) The further handling was carried out analogous to Example 1 G)to J) yielding the Escherichia coli expression strain XL1-pNCO-HIS6-GLY2-SER-GLY-C-Biotag-AA-LuSy.

[0567] G) The further handling was carried out analogous to Example 22G) to H) yielding comparable results.

Example 24

[0568] Construction of a Chimeric Protein Consisting of a Part of theLumazine Synthase from Bacillus subtilis and a Part of the ThermostableLumazine Synthase from Aquifex aeolicus

[0569] A) A part of the gene coding for the lumazine synthase fromBacillus subtilis was amplified analogous to Example 1 A), exceptingthat the oligonucleotide EcoRI-RBS-2 (Example 2 A)) was used as forwardprimer and oligonucleotide BS-LuSy-AgeI (5′ tat tat tat aac cgg tat ttcaaa tgc gcc 3′) was used as reverse primer and the plasmid pNCO-BS-LuSy(see Example 1) was used as template for the PCR. The oligonucleotideBS-LuSy-AgeI was at its 3′-end identical to a region of the ribH genefrom Bacillus subtilis and introduced a recognition site for therestriction endonuclease AgeI (A*CCGGT).

[0570] B) The PCR mixture was analyzed and purified analogous to Example1 B) yielding a DNA-fragment with 225 bp. The purified fragment from B)was digested using the restriction endonuclease AgeI.

[0571] 30.0 μl DNA-fragmente from B)

[0572] 4.0 μl AgeI [8 U]

[0573] 10.0 μl buffer 1(10×) [10 mM Bis-Tris-Propane-HCl, 10 mM MgCl₂, 1mM DTT, pH 7.0]

[0574] 56.0 μl H₂O_(bidest)

[0575] The enzymes was purchased from New England Biolabs (Schwalbach,Germany). The mixture was incubated for 180 min at 25° C. Afterincubation the mixture was purified as described under Example 1 B) andused for the digestion with the restriction endonuclease EcoRI.

[0576] C) In a second step the purified DNA fragment from B) wasdigested with the restriction endonuclease EcoRI.

[0577] 30.0 μl DNA-fragment from B)

[0578] 3.0 μl EcoRI [60 U]

[0579] 20.0 μl OPAU (10×)

[0580] 47.0 μl H₂O_(bidest)

[0581] The enzymes was purchased from New England Biolabs (Schwalbach,Germany). The mixture was incubated for 180 min at 37° C. Afterincubation the mixture was purified as described under Example 1 B).

[0582] D) The plasmid pNCO-AA-LuSy (Example 20, 30 μl, 5 μg) was treatedanalogous to B) and C) and subsequently purified analogous to Example 1B), yielding a DNA-fragment with 3676 bp, which was used in a ligationprotocol.

[0583] E) The further handling was carried out analogous to Example 1 G)to L) yielding the Escherichia coli expression strainXL1-pNCO-BS-LuSy-AgeI-AA-LuSy.

[0584] F) Enzymatic activity could be measured according to Example 1N).

[0585] G) To check the expression rate resp. the molecular weight of thesoluble protein a SDS-PAGE was carried out analogous to Example 1 M). Inthe crude lysate of the strain XL1-pNCO-BS-LuSy-AgeI-AA-LuSy anoverexpressed protein band with a molecular weight of circa 16.4 kDacould be observed which was not detectable in a strain without theplasmid pNCO-BS-LuSy-AgeI-AA-LuSy. The expression rate of this proteincould be estimated to 10% (related to all soluble cell proteins).

Example 25

[0586] Construction of a Vector for the Recombinant N-Terminal Fusion ofTarget Peptides to the Lumazine Synthase from Aquifex aeolicus (TargetPeptides can be Fused Directly without the Use of a Linker Peptide tothe Carrier Protein, whereby the Singular Restriction Site BglII isused, which is Located Inside the Gene Coding for the Carrier Protein)

[0587] A) The gene coding for the lumazine synthase from Aquifexaeolicus was amplified analogous Example 1 A), excepting that theoligonucleotide AQUI-11-BglII (5′ ata ata gaa ttc att aaa gag gag aaatta act atg cag atc tac gaa gg 3′), which bound at its 3′-end to the5′-end of the ribH gene of Aquifex aeolicus and which introduced arecognition site for the restriction endonuclease BglII (A*GATCT) via asilent mutation and which introduced a recognition site for therestriction endonuclease EcoRI (G*AATTC) at the 5′-end, was used asforward primer and oligonucleotide AQUI-10 (see Example 20) was used asreverse primer. The plasmid pNCO-AA-LuSy (Example 20) served as templatefor the PCR.

[0588] B) The PCR mixture was analyzed and purified analogous Example 1B) yielding a DNA-fragment with 510 bp.

[0589] C) The further handling was carried out analogous to Example 1E)-L) yielding the plasmid pNCO-AA-BglII-LuSy.

[0590] D) To check the expression rate resp. the molecular weight of thesoluble protein a SDS-PAGE was carried out analogous to Example 1 M). Inthe crude lysate of the strain XL1-pNCO-AA-BglII-LuSy an overexpressedprotein band with a molecular weight of circa 16.7 kDa could be observedwhich was not detectable in a strain without the plasmidpNCO-AA-BglII-LuSy. The expression rate of this protein could beestimated to 20% (related to all soluble cell proteins).

[0591] E) The further analytics were carried out analogous to Example 20O)-R), whereby no significant difference related to the wild-typeprotein (AA-LuSy) could be observed.

Example 26

[0592] Construction of a Vector for the C-Terminal Fusion of TargetPeptides to the Lumazine Synthase from Aquifex aeolicus

[0593] A) The gene coding for the lumazine synthase from Aquifexaeolicus was amplified analogous Example 1 A), excepting that theoligonucleotide EcoRI-RBS-2 (see Example 2 A)) was used as forwardprimer and oligonucleotide AQUI-10-(BamHI) (5′ tat tat gga tcc tcg gagaga ctt gaa taa gtt tgc 3′), which bound at its 3′-end to the 3′-end ofthe ribH gene from Aquifex aeolicus and which introduced directly afterthe last coding base triplett a recognition site for the restrictionendonuclease BamHI (G*GATCC), whereby the original stop codon wasremoved, was used as reverse primer. The plasmid pNCO-AA-LuSy (Example20) served as template for the PCR.

[0594] B) The PCR mixture was analyzed and purified analogous Example 1B) yielding a DNA-fragment with 507 bp.

[0595] C) The further handling was carried out analogous to Example 1E)-L) yielding the plasmid pNCO-AA-LuSy-(BamHI).

[0596] D) To check the expression rate resp. the molecular weight of thesoluble protein a SDS-PAGE was carried out analogous to Example 1 M). Inthe crude lysate of the strain XL1-pNCO-AA-LuSy-(BamHI) an overexpressedprotein band with a molecular weight of circa 17.8 kDa could be observedwhich was not detectable in a strain without the plasmidpNCO-AA-LuSy-(BamHI). The expression rate of this protein could beestimated to 20% (related to all soluble cell proteins).

[0597] E) The further analytics were carried out analogous to Example 20O)-R), whereby no significant difference related to the wild-typeprotein (AA-LuSy) could be observed.

Example 27

[0598] Construction of a Vector for the Simultaneous N-Terminal andC-Terminal Fusion of Target Peptides to the Lumazine Synthase of Aquifexaeolicus

[0599] A) The gene coding for the lumazine synthase from Aquifexaeolicus was amplified analogous Example 1 A), excepting that theoligonucleotide EcoRI-RBS-2 (see Example 2 A)) was used as forwardprimer and oligonucleotide AQUI-10-(BamHI) (5′ tat tat gga tcc tcg gagaga ctt gaa taa gtt tgc 3′; Example 26) was used as reverse primer andexcepting that the plasmid pNCO-AA-BglII-LuSy (Example 25) served astemplate for the PCR.

[0600] B) The PCR mixture was analyzed and purified analogous Example 1B) yielding a DNA-fragment with 507 bp.

[0601] C) The further handling was carried out analogous to Example 1E)-L) yielding the plasmid pNCO-BglII-AA-LuSy-(BamHI).

[0602] D) To check the expression rate resp. the molecular weight of thesoluble protein a SDS-PAGE was carried out analogous to Example 1 M). Inthe crude lysate of the strain XL1-pNCO-BglII-AA-LuSy-(BamHI) anoverexpressed protein band with a molecular weight of circa 17.8 kDacould be observed which was not detectable in a strain without theplasmid pNCO-AA-BglII-LuSy-(BamHI). The expression rate of this proteincould be estimated to 20% (related to all soluble cell proteins).

[0603] E) The further analytics were carried out analogous to Example 20O)-R), whereby no significant difference related to the wild-typeprotein (AA-LuSy) could be observed.

[0604] Literature

[0605] Altschul, S. F., T. L. Madden, A. A. Schäffer, J. Zhang, Z.Zhang, W. Miller, D. J. Lipman (1997) Gapped BLAST and Psi-BLAST: a newgeneration of protein database search programs Nucleic Acids Res. 25,3389-3402

[0606] Bacher A., R. Ladenstein (1991) The lumazine synthase/riboflavinsynthase complex of Bacillus subtilis in Chemistry and Biochemistry ofFlavoproteins, Band 1 (F. Müller, ed.) CRC Press, Boca Raton, 293-316

[0607] Bacher A., H. C. Ludwig, H. Schnepple and Y. Ben-Shaul (1986)Heavy riboflavin synthase from Bacillus subtilis. Quaternary structureand reaggregation. J. Mol. Biol., 187, 75-86

[0608] Bacher A., H. Schnepple, B. Mailänder, M. K. Otto and Y.Ben-Shaul (1980) Structure and function of the riboflavin synthasecomplex of Bacillus subtilis in Flavins and Flavoproteins, (K. Yagi andT. Yamano, eds.) Japan Scientific Societies Press, 579-586

[0609] Bacher A., S. Eberhardt, M. Fischer, S. Mörtl, K. Kis, K.Kugelbrey, J. Scheuring and K. Schott (1997) Biosynthesis of riboflavin.Lumazine synthase and riboflavin synthase Methods Enzymol., 280, 389-399

[0610] Birnboim H. C., J. Doly (1979) A Rapid Alkaline ExtractionProcedure for Screening Recombinant Plasmid DNA Nucl. Acids. Res., 7,1513-1522

[0611] Bradford M. (1976) A rapid and selective method for thequantitation of microgramm quantities of protein utilizing the prinzipleof protein-dye binding Anal. Biochem., 72, 248-254

[0612] Brigidi P., E. Rossi, M. Bertarini, G. Riccardi, D. Matteuzzi(1990) Genetic transformation of intact cells of Bacillus subtilis byelectroporation FEMS Microbiology Letters, 67, 135-138

[0613] Bullock W. O., J. M. Fernandez, J. M. Short (1987) XL1-Blue: ahigh efficiency plasmid transforming recA Escherichia coli strain withβ-Galactosidase selection BioTechniques, 5, 376-379

[0614] Clackson T., D. Güssow, P. T. Jones (1991) General applicationsof PCR to gene cloning and manipulation In: M. J. McPherson, P. Quirke,G. R. Taylor (eds.); PCR: A practical approach (187-214). Oxford Press

[0615] Cohn E., J. Edsall (1943) Proteins, amino acids and peptides asions and dipolar ions Reinhold, N.Y.

[0616] Compton S. J., C. G. Jones (1985) Mechanism of Dye Response andInterference in the Bradford Protein Assay Annal. Biochem., 151, 369

[0617] Cronan J. E. (1990) Biotination of proteins in vivo The Journalof biological Chemistry, 265/18,10327-10333

[0618] Dalsgaard K., A. Uttenthal, T. Jones, F. Xu, A. Merryweather, W.Hamilton, J. Langeveld, R. Boshuizen, S. Kamstrup, G. Lomonossoff, C.Porta, C. Vela, J. Casal, R. Meloen, P. Rodgers (1997) Plant-derivedvaccine protects target animals against a viral disease NatureBiotechnology, Volume 15, 248-252

[0619] Davis L. G., M. D. Dibner, J. F. Battey (1986) Basic Methods inmolecular biology Elsevier, N.Y., Amsterdam, London

[0620] Deckert G., P. Warren, T. Gaasterland, W. Young, A. Lenox, D.Graham, R. Overbeek, M. Snead, M. Keller, M. Aujay, R. Huber, R.Feldman, J. Short, G. Olsen, R. Swanson (1998) The complete genome ofthe hyperthermophilic bacterium Aquifex aeolicus Nature, 392, 353-358

[0621] Dower W. J., J. F. Miller, C. W. Ragsdale (1988) High efficiencytransformation of Escherichia coli by high voltage electroporation Nucl.Acids Res., 16, 6127-6145

[0622] Glick B. R., J. J. Pasternak (1995) Molekulare BiotechnologieSpektrum Akademischer Verlag, Heidelberg, Berlin, Oxford

[0623] Groseach an H., W. Fiers (1982) Preferential codon usage inprocaryotic genes-the optimal codon-anticodon interaction energy and theselective codon usage in efficiently expressed genes Gene, 18, 199-209

[0624] Hennecke H., I. Günther, F. Binder (1982) A novel cloning vectorfor the direct selection of recombinant DNA in Escherichia coli Gene,19, 231-234

[0625] Henner D. (1990) Expression of Heterologous Genes in Bacillussubtilis Meth. Enzymol., 185, 199

[0626] Ikemura T. (1981) Correlation between the abundance ofEscherichia coli transfer RNAs and the occurence of the respectivecodons in ist protein genes: A proposal for a synonymous codon choicethat is optimal for the Escherichia coli translational system J. Mol.Biol., 151, 389-409

[0627] Ladenstein R., B. Meyer, R. Huber, H. Labischinski, K. Bartels,H.-D. Bartunik, L. Bachmann, H. C. Ludwig and A. Bacher (1986) Heavyriboflavin synthase from Bacillus subtilis. Particle dimensions, crystalpacking and molecular symmetry. J. Mol. Biol., 187, 87-100

[0628] Ladenstein R., H. D. Bartunik, M. Schneider, R. Huber, K. Schottand A. Bacher (1986) Structure of the riboflavin synthase/lumazinesynthase complex. Arrangement and chain folding of 13 subunits in theicosahedral capsid. in Chemistry and Biology of Pteridines, (B. A.Cooper, V. M. Whitehead, eds.) Walter de Gruyter, Berlin, 103-106

[0629] Ladenstein R., K. Ritsert, R. Huber, G. Richter and A. Bacher(1994) The lumazine synthase/riboflavin synthase complex of Bacillussubtilis: X-ray structure analysis of reconstituted β₆₀ capsids at 3.2 Åresolution Eur. J. Biochem., 223, 1007-1017

[0630] Ladenstein R., M. Schneider, R. Huber, H. D. Bartunik, K. Wilson,K. Schott and A. Bacher (1988) Heavy riboflavin synthase from Bacillussubtilis. Crystal structure analysis of the icosahedral β₆₀ capsid at3.3 Å resolution J. Mol. Biol., 203, 1045-1070

[0631] Ladenstein R., M. Schneider, R. Huber, K. Schott and A. Bacher(1988) The structure of the icosahedral β₆₀ capsid of heavy riboflavinsynthase from Bacillus subtilis Z. Kristallographie, 185, 122-124

[0632] Laemmli U. K. (1970) Cleavage of Structural Proteins During theAssembly of the Head of Bacteriophage T4Nature, 227, 680-685

[0633] Laue T., B. Shah, T. Ridgeway, S. Pelletier (1992) Computer aidedinterpretation of analytical sedimentation data for proteins in: HardingS., Rowe A., Horton J. (eds.) Analytical ultracentrifugation inbiochemistry and polymer science Royal society of chemistry, Cambridge,90-125

[0634] LeGrice S. F. J. (1990) A regulated promotor for high levelexpression of heterologous genes in Bacillus subtilis Meth. Enzymol.,185, 201

[0635] Liddell E., I. Weeks (1996) Antikörpertechniken SpektrumAkademischer Verlag, Heidelberg, Berlin, Oxford

[0636] Lottspeich F., H. Zorbas (1998) Bioanalytik Spektrum AkademischerVerlag, Heidelberg, Berlin, Oxford

[0637] Lovett P. S. (1981) BR151ATCC 33677—Bacillus subtilis BR151 J.Bacteriol., 146, 1162-1165

[0638] Maniatis T., E. F. Fritsch, J. Sambrook (1982) Molecular cloning:A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.

[0639] Modrow S., D. Falke (1997) Molekulare Virologie SpektrumAkademischer Veriag, Heidelberg, Berlin, Oxford

[0640] Mörtl S., M. Fischer, G. Richter, J. Tack, S. Weinkauf and A.Bacher (1996) Biosynthesis of riboflavin. Lumazine synthase ofEscherichia coli. J. Biol. Chem., 271, 33201-33207

[0641] Mullis K., F. Faloona, S. Scharf, R. Saiki, G. Horn, H. Ehrlich(1986) Specific enzymatic amplification of DNA in vitro: The polymerasechain reaction Cold Spring Harbor Symp., 51, 263-273

[0642] Perkins J. B., J. G. Pero, A. Sloma (1991) Riboflavinoverproducing bacteria expressing the rib-operon of Bacillus Eur. Pat.Appl. EP 405370 A1910102

[0643] Read S. M., D. H. Northcote (1981) Minimization of variation inthe response to different proteins of the Coomassie Blue G dyebindingassay for protein Anal. Biochem., 116, 53-64

[0644] Sambrook J., E. Fritsch, T. Maniatis (1989) Molecular Cloning: ALaboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

[0645] Sanger F., S. Nicklen, A. R. Coulson (1977) DNA sequencing withchain terminating inhibitors Proc. Natl. Acad. Sci. USA, 74, 5463-5467

[0646] Schatz P. J. (1993) Use of peptide libraries to map the substratespecificity of a peptide-modifying enzyme: a 13 residue consensuspeptide specifies biotinylation in Escherichia coli Bio/Technology,October; 11(10), 1138-1143

[0647] Sgamarella V., J. H. van de Sande, H. G. Khorana (1979) Studieson the polynukleotides. C. A novel joining reaction catalysed by theT4-polynukleotide-ligase Proc. Natl. Acad. Sci. USA, 67, 1468-1475

[0648] Stüber D., H. Matile, G. Garotta (1990) System for high-levelproduction in Escherichia coli and rapid purification of recombinantproteins: Application to epitope mapping, preparation of antibodies andstructure-function analysis in: Lefkovits, I., P. Pernis (eds.)Immunological Methods, IV, 121-152

[0649] Tucker J., Grisshammer (1996) Purification of a rat neurotensinreceptor expressed in Escherichia coli Biochem. Journal, 317, 891-899

[0650] Wada K., Y. Wada, F. Ishibashi, T. Gojobori, T. Ikemura (1992)Codon usage tabulated from the GenBank genetic sequence data. NucleicAcid Res., 20, 2111-2118

[0651] Winnacker E. L. (1990) Gene und Klone. Eine Einführung in dieGentechnologie Verlag Chemie, Weinheim, Dearfield Beach, Basel

[0652] Zamenhof P. J., M. Villarejo (1972) Construction and propertiesof Escherichia coli strains exhibiting α-complementation ofβ-galactosidase fragments in vivo J. Bacteriol., 110, 171-178

[0653]

1 154 1 3420 DNA Artificial sequence pNCO113 Expression vector 1ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca 60attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aaccatggga 120ggatccgtcg acctgcagcc aagcttaatt agctgagctt ggactcctgt tgatagatcc 180agtaatgacc tcagaactcc atctggattt gttcagaacg ctcggttgcc gccgggcgtt 240ttttattggt gagaatccaa gctagcttgg cgagattttc aggagctaag gaagctaaaa 300tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac 360attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata 420ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc 480acattcttgc ccgcctgatg aatgctcatc cggaatttcg tatggcaatg aaagacggtg 540agctggtgat atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa 600cgttttcatc gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt 660cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga 720atatgttttt cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg 780ccaatatgga caacttcttc gcccccgttt tcaccatgca tgggcaaata ttatacgcaa 840ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtctg tgatggcttc 900catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg 960taattttttt aaggcagtta ttggtgccct taaacgcctg gggtaatgac tctctagctt 1020gaggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg 1080tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ctctagagct gcctcgcgcg 1140tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg 1200tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg 1260gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac 1320tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac 1380agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg 1440ctgcgctcgg tctgtcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 1500ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 1560gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 1620gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 1680taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 1740accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc 1800tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 1860cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 1920agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 1980gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 2040gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 2100tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 2160acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 2220cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 2280acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 2340acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 2400tttcgttcat ccatagctgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 2460ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 2520ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 2580tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 2640aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 2700ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 2760ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 2820gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 2880gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 2940cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga 3000actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 3060ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 3120tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 3180ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 3240agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 3300aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc 3360attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcac 3420 25302 DNA Artificial sequence p6021-CAT Expression vector 2 gaattaattcctcgaggctg gcatccctaa catatccgaa tggttactta aacaacggag 60 gactagcgtatcccttcgca tagggtttga gttagataaa gtatatgctg aactttcttc 120 tttgctcaaagaatcataaa aaatttattt gctttcagga aaatttttct gtataataga 180 ttcaaattgtgagcggataa caatttgaat tcattaaaga ggagaaatta actatgaggg 240 gatccgtcgacctgcagcca agcttagcta gctagagctt ggcgagattt tcaggagcta 300 aggaagctaaaatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc 360 atcgtaaagaacattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg 420 ttcagactgcgatgagtggc agggcggggc gtaatttttt taaggcagtt attggtgccc 480 ttaaacgcctggggtaatga ctctctagct tgaggcatca aataaaacga aaggctcagt 540 cgaaagactgggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga 600 caaatccgccgctctagagc tgcctgccgc gtttcggtga tgacggtgaa aacctctgac 660 acatgcagctcccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag 720 cccgtcagggcgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac 780 gtagcgatagcggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag 840 agtgcaccatatgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag 900 gcgctcttccgcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 960 ggtatcagctcactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 1020 aaagaacatgtgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 1080 ggcgtttttccataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 1140 gaggtggcgaaacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 1200 cgtgcgctctcctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 1260 gggaagcgtggcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 1320 tcgctccaagctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 1380 cggtaactatcgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 1440 cactggtaacaggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 1500 gtggcctaactacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc 1560 agttaccttcggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 1620 cggtggtttttttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 1680 tcctttgatcttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 1740 tttggtcatgagattatcaa aaaggatctt cacctagatc cttttcggta ccgctgattt 1800 cactttttgcattctacaaa ctgcataact catatgtaaa tcgctccttt ttaggtggca 1860 caaatgtgaggcattttcgc tctttccggc aaccacttcc aagtaaagta taacacacta 1920 tactttatattcataaagtg tgtgtcctgc gaggcgtcca gtgccgacca aaaccataaa 1980 acctttaagacctttctttt ttttacgaga aaaaagaaac aaaaaaacct gccctctgcc 2040 acctcagcaaaggggggttt tgctctcgtg ctcgtttaaa aatcagcaag ggacaggtag 2100 tattttttgagaagatcact caaaaaatct ccacctttaa acccttgcca atttttattt 2160 tgtccgttttgtctagctta ccgaaagcca gactcagcaa gaataaaatt tttattgtct 2220 ttcggttttctagtgtaacg gacaaaacca ctcaaaataa aaaagataca agagaggtct 2280 ctcgtatcttttattcagca atcgcgcccg attgctgaac agattaataa tagattttag 2340 ctttttatttgttgaaaaaa gctaatcaaa ttgttgtcgg gatcaattac tgcaaagtct 2400 cgttcatcccaccactgatc ttttaatgat gtattggggt gcaaaatgcc caaaggctta 2460 atatgttgatataattcatc aattccctct acttcaatgc ggcaactagc agtaccagca 2520 ataaacgactccgcacctgt acaaaccggt gaatcattac tacgagagcg ccagcttcat 2580 cacttgcctcccatagatga atccgaacct cattacacat tagaactgcg aatccatctt 2640 catggtgaaccaaagtgaaa cctagtttat cgcaataaaa acctatactc tttttaatat 2700 ccccgactggcaatgcggga tagactgtaa cattctcacg cataaaatcc cctttcattt 2760 tctaatgtaaatctattacc ttattattaa ttcaattcgc tcataattaa tcctttttct 2820 tattacgcaaaatggcccga tttaagcaca ccctttattc cgttaatgcg ccatgacagc 2880 catgataattactaatacta ggagaagtta ataaatacgt aaccaacatg attaacaatt 2940 attagaggtcatcgttcaaa atggtatgcg ttttgacaca tccactatat atccgtgtcg 3000 ttctgtccactcctgaatcc cattccagaa attctctagc gattccagaa gtttctcaga 3060 gtcggaaagttgaccagaca ttacgaactg gcacagatgg tcataacctg aaggaagatc 3120 tgattgcttaactgcttcag ttaagaccga agcgctcgtc gtataacaga tgcgatgatg 3180 cagaccaatcaacatggcac ctgccattgc tacctgtaca gtcaaggatg gtagaaatgt 3240 tgtcggtccttgcacacgaa tattacgcca tttgcctgca tattcaaaca gctcttctac 3300 gataagggcacaaatcgcat cgtggaacgt ttgggcttct accgatttag cagttggata 3360 cactttctctaagtatccac ctgaatcata aatcggcaaa atagagaaaa attgaccatg 3420 tgtaagcggccaatctgatt ccacctgaga tgcataatct agtagaatct cttcgctatc 3480 aaaattcacttccaccttcc actcaccggt tgtccattca tggctgaact ctgcttcctc 3540 tgttgacatgacacacatca tctcaatatc cgaatagggc ccatcagtct gacgaccaag 3600 agagccataaacaccaatag ccttaacatc atccccatat ttatccaata ttcgttcctt 3660 aatttcatgaacaatcttca ttctttcttc tctagtcatt attattggtc cattcactat 3720 tctcattcccttttcagata attttagatt tgcttttcta aataagaata tttggagagc 3780 accgttcttattcagctatt aataactcgt cttcctaagc atccttcaat ccttttaata 3840 acaattatagcatctaatct tcaacaaact ggcccgtttg ttgaactact ctttaataaa 3900 ataatttttccgttcccaat tccacattgc aataatagaa aatccatctt catcggcttt 3960 ttcgtcatcatctgtatgaa tcaaatcgcc ttcttctgtg tcatcaaggt ttaatttttt 4020 atgtatttcttttaacaaac caccatagga gattaacctt ttacggtgta aaccttcctc 4080 caaatcagacaaacgtttca aattcttttc ttcatcatcg gtcataaaat ccgtatcctt 4140 tacaggatattttgcagttt cgtcaattgc cgattgtata tccgatttat atttattttt 4200 cggtcgaatcatttgaactt ttacatttgg atcatagtct aatttcattg cctttttcca 4260 aaattgaatccattgttttt gattcacgta gttttctgta ttcttaaaat aagttggttc 4320 cacacataccaatacatgca tgtgctgatt ataagaatta tctttattat ttattgtcac 4380 ttccgttgcacgcataaaac caacaagatt tttattaatt tttttatatt gcatcattcg 4440 gcgaaatccttgagccatat ctgacaaact cttatttaat tcttcgccat cataaacatt 4500 tttaactgttaatgtgagaa acaaccaacg aactgttggc ttttgtttaa taacttcagc 4560 aacaaccttttgtgactgaa tgccatgttt cattgctctc ctccagttgc acattggaca 4620 aagcctggatttacaaaacc acactcgata caactttctt tcgcctgttt cacgattttg 4680 tttatactctaatatttcag cacaatcttt tactctttca gcctttttaa attcaagaat 4740 atgcagaagttcaaagtaat caacattagc gattttcttt tctctccatg gtctcacttt 4800 tccactttttgtcttgtcca ctaaaaccct tgatttttca tctgaataaa tgctactatt 4860 aggacacataatattaaaag aaacccccat ctatttagtt atttgtttag tcacttataa 4920 ctttaacagatggggttttt ctgtgcaacc aattttaagg gttttcaata ctttaaaaca 4980 catacataccaacacttcaa cgcacctttc agcaactaaa ataaaaatga cgttatttct 5040 atatgtatcaagataagaaa gaacaagttc aaaaccatca aaaaaagaca ccttttcagg 5100 tgctttttttattttataaa ctcattccct gatctcgact tcgttctttt tttacctctc 5160 ggttatgagttagttcaaat tcgttctttt taggttctaa atcgtgtttt tcttggaatt 5220 gtgctgttttatcctttacc ttgtctacaa accccttaaa aacgttttta aaggctttta 5280 agccgtctgtacgttcctta ag 5302 3 3885 DNA Artificial sequence pNCO-BS-LuSyExpression vector 3 ctcgagaaat cataaaaaat ttatttgctt tgtgagcggataacaattat aatagattca 60 attgtgagcg gataacaatt tcacacagaa ttcattaaagaggagaaatt aaccatgaat 120 atcatacaag gaaatttagt tggtacaggt cttaaaatcggaatcgtagt aggaagattt 180 aatgatttta ttacgagcaa gctgctgagc ggagcagaagatgcgctgct cagacatggc 240 gtagacacaa atgacattga tgtggcttgg gttccaggcgcatttgaaat accgtttgct 300 gcgaaaaaaa tggcggaaac aaaaaaatat gatgctattatcacattggg cactgtcatc 360 agaggcgcaa cgacacatta cgattatgtc tgcaatgaagctgcaaaagg catcgcgcaa 420 gcagcaaaca ctactggtgt acctgtcatc tttggaattgtaacaactga aaacatcgaa 480 caggctatcg agcgtgccgg cacaaaagcg ggcaacaaaggtgtagattg tgctgtttct 540 gccattgaaa tggcaaactt aaaccgttct ttcgaataaccatggggatc cgtcgacctg 600 cagccaagct taattagctg agcttggact cctgttgatagatccagtaa tgacctcaga 660 actccatctg gatttgttca gaacgctcgg ttgccgccgggcgtttttta ttggtgagaa 720 tccaagctag cttggcgaga ttttcaggag ctaaggaagctaaaatggag aaaaaaatca 780 ctggatatac caccgttgat atatcccaat ggcatcgtaaagaacatttt gaggcatttc 840 agtcagttgc tcaatgtacc tataaccaga ccgttcagctggatattacg gcctttttaa 900 agaccgtaaa gaaaaataag cacaagtttt atccggcctttattcacatt cttgcccgcc 960 tgatgaatgc tcatccggaa tttcgtatgg caatgaaagacggtgagctg gtgatatggg 1020 atagtgttca cccttgttac accgttttcc atgagcaaactgaaacgttt tcatcgctct 1080 ggagtgaata ccacgacgat ttccggcagt ttctacacatatattcgcaa gatgtggcgt 1140 gttacggtga aaacctggcc tatttcccta aagggtttattgagaatatg tttttcgtct 1200 cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaacgtggccaat atggacaact 1260 tcttcgcccc cgttttcacc atgcatgggc aaatattatacgcaaggcga caaggtgctg 1320 atgccgctgg cgattcaggt tcatcatgcc gtctgtgatggcttccatgt cggcagaatg 1380 cttaatgaat tacaacagta ctgcgatgag tggcagggcggggcgtaatt tttttaaggc 1440 agttattggt gcccttaaac gcctggggta atgactctctagcttgaggc atcaaataaa 1500 acgaaaggct cagtcgaaag actgggcctt tcgttttatctgttgtttgt cggtgaacgc 1560 tctcctgagt aggacaaatc cgccgctcta gagctgcctcgcgcgtttcg gtgatgacgg 1620 tgaaaacctc tgacacatgc agctcccgga gacggtcacagcttgtctgt aagcggatgc 1680 cgggagcaga caagcccgtc agggcgcgtc agcgggtgttggcgggtgtc ggggcgcagc 1740 catgacccag tcacgtagcg atagcggagt gtatactggcttaactatgc ggcatcagag 1800 cagattgtac tgagagtgca ccatatgcgg tgtgaaataccgcacagatg cgtaaggaga 1860 aaataccgca tcaggcgctc ttccgcttcc tcgctcactgactcgctgcg ctcggtctgt 1920 cggctgcggc gagcggtatc agctcactca aaggcggtaatacggttatc cacagaatca 1980 ggggataacg caggaaagaa catgtgagca aaaggccagcaaaaggccag gaaccgtaaa 2040 aaggccgcgt tgctggcgtt tttccatagg ctccgcccccctgacgagca tcacaaaaat 2100 cgacgctcaa gtcagaggtg gcgaaacccg acaggactataaagatacca ggcgtttccc 2160 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgccgcttaccgg atacctgtcc 2220 gcctttctcc cttcgggaag cgtggcgctt tctcaatgctcacgctgtag gtatctcagt 2280 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacgaaccccccgt tcagcccgac 2340 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacccggtaagaca cgacttatcg 2400 ccactggcag cagccactgg taacaggatt agcagagcgaggtatgtagg cggtgctaca 2460 gagttcttga agtggtggcc taactacggc tacactagaaggacagtatt tggtatctgc 2520 gctctgctga agccagttac cttcggaaaa agagttggtagctcttgatc cggcaaacaa 2580 accaccgctg gtagcggtgg tttttttgtt tgcaagcagcagattacgcg cagaaaaaaa 2640 ggatctcaag aagatccttt gatcttttct acggggtctgacgctcagtg gaacgaaaac 2700 tcacgttaag ggattttggt catgagatta tcaaaaaggatcttcaccta gatcctttta 2760 aattaaaaat gaagttttaa atcaatctaa agtatatatgagtaaacttg gtctgacagt 2820 taccaatgct taatcagtga ggcacctatc tcagcgatctgtctatttcg ttcatccata 2880 gctgcctgac tccccgtcgt gtagataact acgatacgggagggcttacc atctggcccc 2940 agtgctgcaa tgataccgcg agacccacgc tcaccggctccagatttatc agcaataaac 3000 cagccagccg gaagggccga gcgcagaagt ggtcctgcaactttatccgc ctccatccag 3060 tctattaatt gttgccggga agctagagta agtagttcgccagttaatag tttgcgcaac 3120 gttgttgcca ttgctacagg catcgtggtg tcacgctcgtcgtttggtat ggcttcattc 3180 agctccggtt cccaacgatc aaggcgagtt acatgatcccccatgttgtg caaaaaagcg 3240 gttagctcct tcggtcctcc gatcgttgtc agaagtaagttggccgcagt gttatcactc 3300 atggttatgg cagcactgca taattctctt actgtcatgccatccgtaag atgcttttct 3360 gtgactggtg agtactcaac caagtcattc tgagaatagtgtatgcggcg accgagttgc 3420 tcttgcccgg cgtcaatacg ggataatacc gcgccacatagcagaacttt aaaagtgctc 3480 atcattggaa aacgttcttc ggggcgaaaa ctctcaaggatcttaccgct gttgagatcc 3540 agttcgatgt aacccactcg tgcacccaac tgatcttcagcatcttttac tttcaccagc 3600 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaaaaaagggaat aagggcgaca 3660 cggaaatgtt gaatactcat actcttcctt tttcaatattattgaagcat ttatcagggt 3720 tattgtctca tgagcggata catatttgaa tgtatttagaaaaataaaca aataggggtt 3780 ccgcgcacat ttccccgaaa agtgccacct gacgtctaagaaaccattat tatcatgaca 3840 ttaacctata aaaataggcg tatcacgagg ccctttcgtcttcac 3885 4 5767 DNA Artificial sequence p602-BS-LuSy Expressionplasmid 4 gaattaattc ctcgaggctg gcatccctaa catatccgaa tggttacttaaacaacggag 60 gactagcgta tcccttcgca tagggtttga gttagataaa gtatatgctgaactttcttc 120 tttgctcaaa gaatcataaa aaatttattt gctttcagga aaatttttctgtataataga 180 ttcaaattgt gagcggataa caatttgaat tcattaaaga ggagaaattaactatgaata 240 tcatacaagg aaatttagtt ggtacaggtc ttaaaatcgg aatcgtagtaggaagattta 300 atgattttat tacgagcaag ctgctgagcg gagcagaaga tgcgctgctcagacatggcg 360 tagacacaaa tgacattgat gtggcttggg ttccaggcgc atttgaaataccgtttgctg 420 cgaaaaaaat ggcggaaaca aaaaaatatg atgctattat cacattgggcactgtcatca 480 gaggcgcaac gacacattac gattatgtct gcaatgaagc tgcaaaaggcatcgcgcaag 540 cagcaaacac tactggtgta cctgtcatct ttggaattgt aacaactgaaaacatcgaac 600 aggctatcga gcgtgccggc acaaaagcgg gcaacaaagg tgtagattgtgctgtttctg 660 ccattgaaat ggcaaactta aaccgttctt tcgaataacc atggggatccgtcgacctgc 720 agccaagctt agctagctag agcttggcga gattttcagg agctaaggaagctaaaatgg 780 agaaaaaaat cactggatat accaccgttg atatatccca atggcatcgtaaagaacatt 840 ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcagactgcgatga 900 gtggcagggc ggggcgtaat ttttttaagg cagttattgg tgcccttaaacgcctggggt 960 aatgactctc tagcttgagg catcaaataa aacgaaaggc tcagtcgaaagactgggcct 1020 ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaatccgccgctct 1080 agagctgcct gccgcgtttc ggtgatgacg gtgaaaacct ctgacacatgcagctcccgg 1140 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgtcagggcgcgt 1200 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagcgatagcggag 1260 tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgcaccatatgcg 1320 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgctcttccgcttc 1380 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtatcagctcactc 1440 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaagaacatgtgagc 1500 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgtttttccatag 1560 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggtggcgaaaccc 1620 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgcgctctcctgt 1680 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaagcgtggcgct 1740 ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgctccaagctggg 1800 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggtaactatcgtct 1860 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactggtaacaggat 1920 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggcctaactacgg 1980 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagttaccttcggaaa 2040 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtggtttttttgt 2100 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctttgatcttttc 2160 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttggtcatgagatt 2220 atcaaaaagg atcttcacct agatcctttt cggtaccgct gatttcactttttgcattct 2280 acaaactgca taactcatat gtaaatcgct cctttttagg tggcacaaatgtgaggcatt 2340 ttcgctcttt ccggcaacca cttccaagta aagtataaca cactatactttatattcata 2400 aagtgtgtgt cctgcgaggc gtccagtgcc gaccaaaacc ataaaacctttaagaccttt 2460 ctttttttta cgagaaaaaa gaaacaaaaa aacctgccct ctgccacctcagcaaagggg 2520 ggttttgctc tcgtgctcgt ttaaaaatca gcaagggaca ggtagtattttttgagaaga 2580 tcactcaaaa aatctccacc tttaaaccct tgccaatttt tattttgtccgttttgtcta 2640 gcttaccgaa agccagactc agcaagaata aaatttttat tgtctttcggttttctagtg 2700 taacggacaa aaccactcaa aataaaaaag atacaagaga ggtctctcgtatcttttatt 2760 cagcaatcgc gcccgattgc tgaacagatt aataatagat tttagctttttatttgttga 2820 aaaaagctaa tcaaattgtt gtcgggatca attactgcaa agtctcgttcatcccaccac 2880 tgatctttta atgatgtatt ggggtgcaaa atgcccaaag gcttaatatgttgatataat 2940 tcatcaattc cctctacttc aatgcggcaa ctagcagtac cagcaataaacgactccgca 3000 cctgtacaaa ccggtgaatc attactacga gagcgccagc ttcatcacttgcctcccata 3060 gatgaatccg aacctcatta cacattagaa ctgcgaatcc atcttcatggtgaaccaaag 3120 tgaaacctag tttatcgcaa taaaaaccta tactcttttt aatatccccgactggcaatg 3180 cgggatagac tgtaacattc tcacgcataa aatccccttt cattttctaatgtaaatcta 3240 ttaccttatt attaattcaa ttcgctcata attaatcctt tttcttattacgcaaaatgg 3300 cccgatttaa gcacaccctt tattccgtta atgcgccatg acagccatgataattactaa 3360 tactaggaga agttaataaa tacgtaacca acatgattaa caattattagaggtcatcgt 3420 tcaaaatggt atgcgttttg acacatccac tatatatccg tgtcgttctgtccactcctg 3480 aatcccattc cagaaattct ctagcgattc cagaagtttc tcagagtcggaaagttgacc 3540 agacattacg aactggcaca gatggtcata acctgaagga agatctgattgcttaactgc 3600 ttcagttaag accgaagcgc tcgtcgtata acagatgcga tgatgcagaccaatcaacat 3660 ggcacctgcc attgctacct gtacagtcaa ggatggtaga aatgttgtcggtccttgcac 3720 acgaatatta cgccatttgc ctgcatattc aaacagctct tctacgataagggcacaaat 3780 cgcatcgtgg aacgtttggg cttctaccga tttagcagtt ggatacactttctctaagta 3840 tccacctgaa tcataaatcg gcaaaataga gaaaaattga ccatgtgtaagcggccaatc 3900 tgattccacc tgagatgcat aatctagtag aatctcttcg ctatcaaaattcacttccac 3960 cttccactca ccggttgtcc attcatggct gaactctgct tcctctgttgacatgacaca 4020 catcatctca atatccgaat agggcccatc agtctgacga ccaagagagccataaacacc 4080 aatagcctta acatcatccc catatttatc caatattcgt tccttaatttcatgaacaat 4140 cttcattctt tcttctctag tcattattat tggtccattc actattctcattcccttttc 4200 agataatttt agatttgctt ttctaaataa gaatatttgg agagcaccgttcttattcag 4260 ctattaataa ctcgtcttcc taagcatcct tcaatccttt taataacaattatagcatct 4320 aatcttcaac aaactggccc gtttgttgaa ctactcttta ataaaataatttttccgttc 4380 ccaattccac attgcaataa tagaaaatcc atcttcatcg gctttttcgtcatcatctgt 4440 atgaatcaaa tcgccttctt ctgtgtcatc aaggtttaat tttttatgtatttcttttaa 4500 caaaccacca taggagatta accttttacg gtgtaaacct tcctccaaatcagacaaacg 4560 tttcaaattc ttttcttcat catcggtcat aaaatccgta tcctttacaggatattttgc 4620 agtttcgtca attgccgatt gtatatccga tttatattta tttttcggtcgaatcatttg 4680 aacttttaca tttggatcat agtctaattt cattgccttt ttccaaaattgaatccattg 4740 tttttgattc acgtagtttt ctgtattctt aaaataagtt ggttccacacataccaatac 4800 atgcatgtgc tgattataag aattatcttt attatttatt gtcacttccgttgcacgcat 4860 aaaaccaaca agatttttat taattttttt atattgcatc attcggcgaaatccttgagc 4920 catatctgac aaactcttat ttaattcttc gccatcataa acatttttaactgttaatgt 4980 gagaaacaac caacgaactg ttggcttttg tttaataact tcagcaacaaccttttgtga 5040 ctgaatgcca tgtttcattg ctctcctcca gttgcacatt ggacaaagcctggatttaca 5100 aaaccacact cgatacaact ttctttcgcc tgtttcacga ttttgtttatactctaatat 5160 ttcagcacaa tcttttactc tttcagcctt tttaaattca agaatatgcagaagttcaaa 5220 gtaatcaaca ttagcgattt tcttttctct ccatggtctc acttttccactttttgtctt 5280 gtccactaaa acccttgatt tttcatctga ataaatgcta ctattaggacacataatatt 5340 aaaagaaacc cccatctatt tagttatttg tttagtcact tataactttaacagatgggg 5400 tttttctgtg caaccaattt taagggtttt caatacttta aaacacatacataccaacac 5460 ttcaacgcac ctttcagcaa ctaaaataaa aatgacgtta tttctatatgtatcaagata 5520 agaaagaaca agttcaaaac catcaaaaaa agacaccttt tcaggtgctttttttatttt 5580 ataaactcat tccctgatct cgacttcgtt ctttttttac ctctcggttatgagttagtt 5640 caaattcgtt ctttttaggt tctaaatcgt gtttttcttg gaattgtgctgttttatcct 5700 ttaccttgtc tacaaacccc ttaaaaacgt ttttaaaggc ttttaagccgtctgtacgtt 5760 ccttaag 5767 5 3879 DNA Artificial sequencepNCO-N-BS-LuSy-C93S Expression plasmid 5 ctcgagaaat cataaaaaatttatttgctt tgtgagcgga taacaattat aatagattca 60 attgtgagcg gataacaatttcacacagaa ttcattaaag aggagaaatt aaccatgaat 120 atcatacaag gaaatttagttggtacaggt cttaaaatcg gaatcgtagt aggaagattt 180 aatgatttta ttacgagcaagctgctgagc ggagcagaag atgcgctgct cagacatggc 240 gtagacacaa atgacattgatgtggcttgg gttccaggcg catttgaaat accgtttgct 300 gcgaaaaaaa tggcggaaacaaaaaaatat gatgctatta tcacattggg cactgtcatc 360 agaggcgcaa cgacacattacgattatgtt tcgaatgaag ctgcaaaagg catcgcgcaa 420 gcagcaaaca ctactggtgtacctgtcatc tttggaattg taacaactga aaacatcgaa 480 caggctatcg agcgtgccggcacaaaagcg ggcaacaaag gtgtagattg tgctgtttct 540 gccattgaaa tggcaaatttaaaccgctca tttgaataag gatccgtcga cctgcagcca 600 agcttaatta gctgagcttggactcctgtt gatagatcca gtaatgacct cagaactcca 660 tctggatttg ttcagaacgctcggttgccg ccgggcgttt tttattggtg agaatccaag 720 ctagcttggc gagattttcaggagctaagg aagctaaaat ggagaaaaaa atcactggat 780 ataccaccgt tgatatatcccaatggcatc gtaaagaaca ttttgaggca tttcagtcag 840 ttgctcaatg tacctataaccagaccgttc agctggatat tacggccttt ttaaagaccg 900 taaagaaaaa taagcacaagttttatccgg cctttattca cattcttgcc cgcctgatga 960 atgctcatcc ggaatttcgtatggcaatga aagacggtga gctggtgata tgggatagtg 1020 ttcacccttg ttacaccgttttccatgagc aaactgaaac gttttcatcg ctctggagtg 1080 aataccacga cgatttccggcagtttctac acatatattc gcaagatgtg gcgtgttacg 1140 gtgaaaacct ggcctatttccctaaagggt ttattgagaa tatgtttttc gtctcagcca 1200 atccctgggt gagtttcaccagttttgatt taaacgtggc caatatggac aacttcttcg 1260 cccccgtttt caccatgcatgggcaaatat tatacgcaag gcgacaaggt gctgatgccg 1320 ctggcgattc aggttcatcatgccgtctgt gatggcttcc atgtcggcag aatgcttaat 1380 gaattacaac agtactgcgatgagtggcag ggcggggcgt aattttttta aggcagttat 1440 tggtgccctt aaacgcctggggtaatgact ctctagcttg aggcatcaaa taaaacgaaa 1500 ggctcagtcg aaagactgggcctttcgttt tatctgttgt ttgtcggtga acgctctcct 1560 gagtaggaca aatccgccgctctagagctg cctcgcgcgt ttcggtgatg acggtgaaaa 1620 cctctgacac atgcagctcccggagacggt cacagcttgt ctgtaagcgg atgccgggag 1680 cagacaagcc cgtcagggcgcgtcagcggg tgttggcggg tgtcggggcg cagccatgac 1740 ccagtcacgt agcgatagcggagtgtatac tggcttaact atgcggcatc agagcagatt 1800 gtactgagag tgcaccatatgcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac 1860 cgcatcaggc gctcttccgcttcctcgctc actgactcgc tgcgctcggt ctgtcggctg 1920 cggcgagcgg tatcagctcactcaaaggcg gtaatacggt tatccacaga atcaggggat 1980 aacgcaggaa agaacatgtgagcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 2040 gcgttgctgg cgtttttccataggctccgc ccccctgacg agcatcacaa aaatcgacgc 2100 tcaagtcaga ggtggcgaaacccgacagga ctataaagat accaggcgtt tccccctgga 2160 agctccctcg tgcgctctcctgttccgacc ctgccgctta ccggatacct gtccgccttt 2220 ctcccttcgg gaagcgtggcgctttctcaa tgctcacgct gtaggtatct cagttcggtg 2280 taggtcgttc gctccaagctgggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 2340 gccttatccg gtaactatcgtcttgagtcc aacccggtaa gacacgactt atcgccactg 2400 gcagcagcca ctggtaacaggattagcaga gcgaggtatg taggcggtgc tacagagttc 2460 ttgaagtggt ggcctaactacggctacact agaaggacag tatttggtat ctgcgctctg 2520 ctgaagccag ttaccttcggaaaaagagtt ggtagctctt gatccggcaa acaaaccacc 2580 gctggtagcg gtggtttttttgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 2640 caagaagatc ctttgatcttttctacgggg tctgacgctc agtggaacga aaactcacgt 2700 taagggattt tggtcatgagattatcaaaa aggatcttca cctagatcct tttaaattaa 2760 aaatgaagtt ttaaatcaatctaaagtata tatgagtaaa cttggtctga cagttaccaa 2820 tgcttaatca gtgaggcacctatctcagcg atctgtctat ttcgttcatc catagctgcc 2880 tgactccccg tcgtgtagataactacgata cgggagggct taccatctgg ccccagtgct 2940 gcaatgatac cgcgagacccacgctcaccg gctccagatt tatcagcaat aaaccagcca 3000 gccggaaggg ccgagcgcagaagtggtcct gcaactttat ccgcctccat ccagtctatt 3060 aattgttgcc gggaagctagagtaagtagt tcgccagtta atagtttgcg caacgttgtt 3120 gccattgcta caggcatcgtggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 3180 ggttcccaac gatcaaggcgagttacatga tcccccatgt tgtgcaaaaa agcggttagc 3240 tccttcggtc ctccgatcgttgtcagaagt aagttggccg cagtgttatc actcatggtt 3300 atggcagcac tgcataattctcttactgtc atgccatccg taagatgctt ttctgtgact 3360 ggtgagtact caaccaagtcattctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 3420 ccggcgtcaa tacgggataataccgcgcca catagcagaa ctttaaaagt gctcatcatt 3480 ggaaaacgtt cttcggggcgaaaactctca aggatcttac cgctgttgag atccagttcg 3540 atgtaaccca ctcgtgcacccaactgatct tcagcatctt ttactttcac cagcgtttct 3600 gggtgagcaa aaacaggaaggcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 3660 tgttgaatac tcatactcttcctttttcaa tattattgaa gcatttatca gggttattgt 3720 ctcatgagcg gatacatatttgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 3780 acatttcccc gaaaagtgccacctgacgtc taagaaacca ttattatcat gacattaacc 3840 tataaaaata ggcgtatcacgaggcccttt cgtcttcac 3879 6 3879 DNA Artificial sequence pNCO-C-BS-LuSyC139S Expression plasmid 6 ctcgagaaat cataaaaaat ttatttgctt tgtgagcggataacaattat aatagattca 60 attgtgagcg gataacaatt tcacacagaa ttcattaaagaggagaaatt aaccatgaat 120 atcatacaag gaaatttagt tggtacaggt cttaaaatcggaatcgtagt aggaagattt 180 aatgatttta ttacgagcaa gctgctgagc ggagcagaagatgcgctgct cagacatggc 240 gtagacacaa atgacattga tgtggcttgg gttccaggcgcatttgaaat accgtttgct 300 gcgaaaaaaa tggcggaaac aaaaaaatat gatgctattatcacattggg cactgtcatc 360 agaggcgcaa cgacacatta cgattatgtc tgcaatgaagctgcaaaagg catcgcgcaa 420 gcagcaaaca ctactggtgt acctgtcatc tttggaattgtaacaactga aaacatcgaa 480 caggctatcg agcgtgccgg cacaaaagcg ggcaacaaaggtgtagattc agctgtttct 540 gccattgaaa tggcaaattt aaaccgctca tttgaataaggatccgtcga cctgcagcca 600 agcttaatta gctgagcttg gactcctgtt gatagatccagtaatgacct cagaactcca 660 tctggatttg ttcagaacgc tcggttgccg ccgggcgttttttattggtg agaatccaag 720 ctagcttggc gagattttca ggagctaagg aagctaaaatggagaaaaaa atcactggat 780 ataccaccgt tgatatatcc caatggcatc gtaaagaacattttgaggca tttcagtcag 840 ttgctcaatg tacctataac cagaccgttc agctggatattacggccttt ttaaagaccg 900 taaagaaaaa taagcacaag ttttatccgg cctttattcacattcttgcc cgcctgatga 960 atgctcatcc ggaatttcgt atggcaatga aagacggtgagctggtgata tgggatagtg 1020 ttcacccttg ttacaccgtt ttccatgagc aaactgaaacgttttcatcg ctctggagtg 1080 aataccacga cgatttccgg cagtttctac acatatattcgcaagatgtg gcgtgttacg 1140 gtgaaaacct ggcctatttc cctaaagggt ttattgagaatatgtttttc gtctcagcca 1200 atccctgggt gagtttcacc agttttgatt taaacgtggccaatatggac aacttcttcg 1260 cccccgtttt caccatgcat gggcaaatat tatacgcaaggcgacaaggt gctgatgccg 1320 ctggcgattc aggttcatca tgccgtctgt gatggcttccatgtcggcag aatgcttaat 1380 gaattacaac agtactgcga tgagtggcag ggcggggcgtaattttttta aggcagttat 1440 tggtgccctt aaacgcctgg ggtaatgact ctctagcttgaggcatcaaa taaaacgaaa 1500 ggctcagtcg aaagactggg cctttcgttt tatctgttgtttgtcggtga acgctctcct 1560 gagtaggaca aatccgccgc tctagagctg cctcgcgcgtttcggtgatg acggtgaaaa 1620 cctctgacac atgcagctcc cggagacggt cacagcttgtctgtaagcgg atgccgggag 1680 cagacaagcc cgtcagggcg cgtcagcggg tgttggcgggtgtcggggcg cagccatgac 1740 ccagtcacgt agcgatagcg gagtgtatac tggcttaactatgcggcatc agagcagatt 1800 gtactgagag tgcaccatat gcggtgtgaa ataccgcacagatgcgtaag gagaaaatac 1860 cgcatcaggc gctcttccgc ttcctcgctc actgactcgctgcgctcggt ctgtcggctg 1920 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggttatccacaga atcaggggat 1980 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaaggccaggaaccg taaaaaggcc 2040 gcgttgctgg cgtttttcca taggctccgc ccccctgacgagcatcacaa aaatcgacgc 2100 tcaagtcaga ggtggcgaaa cccgacagga ctataaagataccaggcgtt tccccctgga 2160 agctccctcg tgcgctctcc tgttccgacc ctgccgcttaccggatacct gtccgccttt 2220 ctcccttcgg gaagcgtggc gctttctcaa tgctcacgctgtaggtatct cagttcggtg 2280 taggtcgttc gctccaagct gggctgtgtg cacgaaccccccgttcagcc cgaccgctgc 2340 gccttatccg gtaactatcg tcttgagtcc aacccggtaagacacgactt atcgccactg 2400 gcagcagcca ctggtaacag gattagcaga gcgaggtatgtaggcggtgc tacagagttc 2460 ttgaagtggt ggcctaacta cggctacact agaaggacagtatttggtat ctgcgctctg 2520 ctgaagccag ttaccttcgg aaaaagagtt ggtagctcttgatccggcaa acaaaccacc 2580 gctggtagcg gtggtttttt tgtttgcaag cagcagattacgcgcagaaa aaaaggatct 2640 caagaagatc ctttgatctt ttctacgggg tctgacgctcagtggaacga aaactcacgt 2700 taagggattt tggtcatgag attatcaaaa aggatcttcacctagatcct tttaaattaa 2760 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaacttggtctga cagttaccaa 2820 tgcttaatca gtgaggcacc tatctcagcg atctgtctatttcgttcatc catagctgcc 2880 tgactccccg tcgtgtagat aactacgata cgggagggcttaccatctgg ccccagtgct 2940 gcaatgatac cgcgagaccc acgctcaccg gctccagatttatcagcaat aaaccagcca 3000 gccggaaggg ccgagcgcag aagtggtcct gcaactttatccgcctccat ccagtctatt 3060 aattgttgcc gggaagctag agtaagtagt tcgccagttaatagtttgcg caacgttgtt 3120 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttggtatggcttc attcagctcc 3180 ggttcccaac gatcaaggcg agttacatga tcccccatgttgtgcaaaaa agcggttagc 3240 tccttcggtc ctccgatcgt tgtcagaagt aagttggccgcagtgttatc actcatggtt 3300 atggcagcac tgcataattc tcttactgtc atgccatccgtaagatgctt ttctgtgact 3360 ggtgagtact caaccaagtc attctgagaa tagtgtatgcggcgaccgag ttgctcttgc 3420 ccggcgtcaa tacgggataa taccgcgcca catagcagaactttaaaagt gctcatcatt 3480 ggaaaacgtt cttcggggcg aaaactctca aggatcttaccgctgttgag atccagttcg 3540 atgtaaccca ctcgtgcacc caactgatct tcagcatcttttactttcac cagcgtttct 3600 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagggaataagggc gacacggaaa 3660 tgttgaatac tcatactctt cctttttcaa tattattgaagcatttatca gggttattgt 3720 ctcatgagcg gatacatatt tgaatgtatt tagaaaaataaacaaatagg ggttccgcgc 3780 acatttcccc gaaaagtgcc acctgacgtc taagaaaccattattatcat gacattaacc 3840 tataaaaata ggcgtatcac gaggcccttt cgtcttcac3879 7 3879 DNA Artificial sequence pNCO-BS-LuSy C93/139S Expressionplasmid 7 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattataatagattca 60 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaattaaccatgaat 120 atcatacaag gaaatttagt tggtacaggt cttaaaatcg gaatcgtagtaggaagattt 180 aatgatttta ttacgagcaa gctgctgagc ggagcagaag atgcgctgctcagacatggc 240 gtagacacaa atgacattga tgtggcttgg gttccaggcg catttgaaataccgtttgct 300 gcgaaaaaaa tggcggaaac aaaaaaatat gatgctatta tcacattgggcactgtcatc 360 agaggcgcaa cgacacatta cgattatgtt tcgaatgaag ctgcaaaaggcatcgcgcaa 420 gcagcaaaca ctactggtgt acctgtcatc tttggaattg taacaactgaaaacatcgaa 480 caggctatcg agcgtgccgg cacaaaagcg ggcaacaaag gtgtagattcagctgtttct 540 gccattgaaa tggcaaattt aaaccgctca tttgaataag gatccgtcgacctgcagcca 600 agcttaatta gctgagcttg gactcctgtt gatagatcca gtaatgacctcagaactcca 660 tctggatttg ttcagaacgc tcggttgccg ccgggcgttt tttattggtgagaatccaag 720 ctagcttggc gagattttca ggagctaagg aagctaaaat ggagaaaaaaatcactggat 780 ataccaccgt tgatatatcc caatggcatc gtaaagaaca ttttgaggcatttcagtcag 840 ttgctcaatg tacctataac cagaccgttc agctggatat tacggcctttttaaagaccg 900 taaagaaaaa taagcacaag ttttatccgg cctttattca cattcttgcccgcctgatga 960 atgctcatcc ggaatttcgt atggcaatga aagacggtga gctggtgatatgggatagtg 1020 ttcacccttg ttacaccgtt ttccatgagc aaactgaaac gttttcatcgctctggagtg 1080 aataccacga cgatttccgg cagtttctac acatatattc gcaagatgtggcgtgttacg 1140 gtgaaaacct ggcctatttc cctaaagggt ttattgagaa tatgtttttcgtctcagcca 1200 atccctgggt gagtttcacc agttttgatt taaacgtggc caatatggacaacttcttcg 1260 cccccgtttt caccatgcat gggcaaatat tatacgcaag gcgacaaggtgctgatgccg 1320 ctggcgattc aggttcatca tgccgtctgt gatggcttcc atgtcggcagaatgcttaat 1380 gaattacaac agtactgcga tgagtggcag ggcggggcgt aatttttttaaggcagttat 1440 tggtgccctt aaacgcctgg ggtaatgact ctctagcttg aggcatcaaataaaacgaaa 1500 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtgaacgctctcct 1560 gagtaggaca aatccgccgc tctagagctg cctcgcgcgt ttcggtgatgacggtgaaaa 1620 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcggatgccgggag 1680 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcgcagccatgac 1740 ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatcagagcagatt 1800 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaaggagaaaatac 1860 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggtctgtcggctg 1920 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacagaatcaggggat 1980 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccgtaaaaaggcc 2040 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaaaaatcgacgc 2100 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtttccccctgga 2160 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacctgtccgccttt 2220 ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatctcagttcggtg 2280 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcccgaccgctgc 2340 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgacttatcgccactg 2400 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgctacagagttc 2460 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtatctgcgctctg 2520 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaaacaaaccacc 2580 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaaaaaaggatct 2640 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacgaaaactcacgt 2700 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatccttttaaattaa 2760 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctgacagttaccaa 2820 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatccatagctgcc 2880 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctggccccagtgct 2940 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaataaaccagcca 3000 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccatccagtctatt 3060 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcgcaacgttgtt 3120 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttcattcagctcc 3180 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaaagcggttagc 3240 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatcactcatggtt 3300 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgcttttctgtgact 3360 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgagttgctcttgc 3420 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagtgctcatcatt 3480 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgagatccagttcg 3540 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcaccagcgtttct 3600 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggcgacacggaaa 3660 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatcagggttattgt 3720 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaataggggttccgcgc 3780 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcatgacattaacc 3840 tataaaaata ggcgtatcac gaggcccttt cgtcttcac 3879 8 3912DNA Artificial sequence pNCO-EC-N-BS-LuSy Expression vector 8 ctcgagaaatcataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca 60 attgtgagcggataacaatt tcacacagaa ttcattaaag aggagaaatt aactatggcg 120 gcggcgcgtagctgcgcggc cgctatgaat atcatacaag gaaatttagt tggtacaggt 180 cttaaaatcggaatcgtagt aggaagattt aatgatttta ttacgagcaa gctgctgagc 240 ggagcagaagatgcgctgct cagacatggc gtagacacaa atgacattga tgtggcttgg 300 gttccaggcgcatttgaaat accgtttgct gcgaaaaaaa tggcggaaac aaaaaaatat 360 gatgctattatcacattggg cactgtcatc agaggcgcaa cgacacatta cgattatgtc 420 tgcaatgaagctgcaaaagg catcgcgcaa gcagcaaaca ctactggtgt acctgtcatc 480 tttggaattgtaacaactga aaacatcgaa caggctatcg agcgtgccgg cacaaaagcg 540 ggcaacaaaggtgtagattg tgctgtttct gccattgaaa tggcaaattt aaaccgctca 600 tttgaataatttggatccgt cgacctgcag ccaagcttaa ttagctgagc ttggactcct 660 gttgatagatccagtaatga cctcagaact ccatctggat ttgttcagaa cgctcggttg 720 ccgccgggcgttttttattg gtgagaatcc aagctagctt ggcgagattt tcaggagcta 780 aggaagctaaaatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc 840 atcgtaaagaacattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg 900 ttcagctggatattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc 960 cggcctttattcacattctt gcccgcctga tgaatgctca tccggaattt cgtatggcaa 1020 tgaaagacggtgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg 1080 agcaaactgaaacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc 1140 tacacatatattcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag 1200 ggtttattgagaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg 1260 atttaaacgtggccaatatg gacaacttct tcgcccccgt tttcaccatg catgggcaaa 1320 tattatacgcaaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtc 1380 tgtgatggcttccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg 1440 cagggcggggcgtaattttt ttaaggcagt tattggtgcc cttaaacgcc tggggtaatg 1500 actctctagcttgaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg 1560 ttttatctgttgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgctctagag 1620 ctgcctcgcgcgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac 1680 ggtcacagcttgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc 1740 gggtgttggcgggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta 1800 tactggcttaactatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt 1860 gaaataccgcacagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg 1920 ctcactgactcgctgcgctc ggtctgtcgg ctgcggcgag cggtatcagc tcactcaaag 1980 gcggtaatacggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 2040 ggccagcaaaaggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 2100 cgcccccctgacgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 2160 ggactataaagataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 2220 accctgccgcttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 2280 caatgctcacgctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 2340 gtgcacgaaccccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 2400 tccaacccggtaagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 2460 agagcgaggtatgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 2520 actagaaggacagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 2580 gttggtagctcttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 2640 aagcagcagattacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 2700 gggtctgacgctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 2760 aaaaggatcttcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 2820 atatatgagtaaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 2880 gcgatctgtctatttcgttc atccatagct gcctgactcc ccgtcgtgta gataactacg 2940 atacgggagggcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 3000 ccggctccagatttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 3060 cctgcaactttatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 3120 agttcgccagttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 3180 cgctcgtcgtttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 3240 tgatcccccatgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 3300 agtaagttggccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 3360 gtcatgccatccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 3420 gaatagtgtatgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 3480 ccacatagcagaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 3540 tcaaggatcttaccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 3600 tcttcagcatcttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 3660 gccgcaaaaaagggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 3720 caatattattgaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 3780 atttagaaaaataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 3840 gtctaagaaaccattattat catgacatta acctataaaa ataggcgtat cacgaggccc 3900 tttcgtcttcac 3912 9 3900 DNA Artificial sequence pNCO-C-BS-LuSy Expression vector9 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca 60attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaat 120atcatacaag gaaatttagt tggtacaggt cttaaaatcg gaatcgtagt aggaagattt 180aatgatttta ttacgagcaa gctgctgagc ggagcagaag atgcgctgct cagacatggc 240gtagacacaa atgacattga tgtggcttgg gttccaggcg catttgaaat accgtttgct 300gcgaaaaaaa tggcggaaac aaaaaaatat gatgctatta tcacattggg cactgtcatc 360agaggcgcaa cgacacatta cgattatgtc tgcaatgaag ctgcaaaagg catcgcgcaa 420gcagcaaaca ctactggtgt acctgtcatc tttggaattg taacaactga aaacatcgaa 480caggctatcg agcgtgccgg cacaaaagcg ggcaacaaag gtgtagattg tgctgtttct 540gccattgaaa tggcaaattt aaaccgctca tttgaattag cggccgcaaa cagtttaaaa 600ggatccgtcg acctgcagcc aagcttaatt agctgagctt ggactcctgt tgatagatcc 660agtaatgacc tcagaactcc atctggattt gttcagaacg ctcggttgcc gccgggcgtt 720ttttattggt gagaatccaa gctagcttgg cgagattttc aggagctaag gaagctaaaa 780tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac 840attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata 900ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc 960acattcttgc ccgcctgatg aatgctcatc cggaatttcg tatggcaatg aaagacggtg 1020agctggtgat atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa 1080cgttttcatc gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt 1140cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga 1200atatgttttt cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg 1260ccaatatgga caacttcttc gcccccgttt tcaccatgca tgggcaaata ttatacgcaa 1320ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtctg tgatggcttc 1380catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg 1440taattttttt aaggcagtta ttggtgccct taaacgcctg gggtaatgac tctctagctt 1500gaggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg 1560tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ctctagagct gcctcgcgcg 1620tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg 1680tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg 1740gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac 1800tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac 1860agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg 1920ctgcgctcgg tctgtcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 1980ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 2040gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 2100gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 2160taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 2220accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc 2280tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 2340cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 2400agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 2460gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 2520gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 2580tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 2640acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 2700cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 2760acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 2820acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 2880tttcgttcat ccatagctgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 2940ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 3000ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 3060tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 3120aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 3180ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 3240ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 3300gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 3360gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 3420cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga 3480actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 3540ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 3600tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 3660ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 3720agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 3780aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc 3840attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcac 390010 4368 DNA Artificial sequence pNCO-EC-DHFR-BS-LuSy Expression vector10 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca 60attgtgagcg gataacaatt tcacacagaa ttgattaaag aggagaaatt aactatgatc 120agtctgattg cggcgttagc ggtagatcgc gttatcggca tggaaaacgc catgccgtgg 180aacctgcctg ccgatctcgc ctggtttaaa cgcaacacct taaataaacc cgtgattatg 240ggccgccata cctgggaatc aatcggtcgt ccgttgccag gacgcaaaaa tattatcctc 300agcagtcaac cgggtacgga cgatcgcgta acgtgggtga agtcggtgga tgaagccatc 360gcggcgtgtg gtgacgtacc agaaatcatg gtgattggcg gcggtcgcgt ttatgaacag 420ttcttgccaa aagcgcaaaa actgtatctg acgcatatcg acgcagaagt ggaaggcgac 480acccatttcc cggattacga gccggatgac tgggaatcgg tattcagcga attccacgat 540gctgatgcgc agaactctca cagctattgc tttgagattc tggagcggcg tgcggccgct 600atgaatatca tacaaggaaa tttagttggt acaggtctta aaatcggaat cgtagtagga 660agatttaatg attttattac gagcaagctg ctgagcggag cagaagatgc gctgctcaga 720catggcgtag acacaaatga cattgatgtg gcttgggttc caggcgcatt tgaaataccg 780tttgctgcga aaaaaatggc ggaaacaaaa aaatatgatg ctattatcac attgggcact 840gtcatcagag gcgcaacgac acattacgat tatgtctgca atgaagctgc aaaaggcatc 900gcgcaagcag caaacactac tggtgtacct gtcatctttg gaattgtaac aactgaaaac 960atcgaacagg ctatcgagcg tgccggcaca aaagcgggca acaaaggtgt agattgtgct 1020gtttctgcca ttgaaatggc aaatttaaac cgctcatttg aataatttgg atccgtcgac 1080ctgcagccaa gcttaattag ctgagcttgg actcctgttg atagatccag taatgacctc 1140agaactccat ctggatttgt tcagaacgct cggttgccgc cgggcgtttt ttattggtga 1200gaatccaagc tagcttggcg agattttcag gagctaagga agctaaaatg gagaaaaaaa 1260tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat 1320ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt 1380taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc 1440gcctgatgaa tgctcatccg gaatttcgta tggcaatgaa agacggtgag ctggtgatat 1500gggatagtgt tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc 1560tctggagtga ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg 1620cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg 1680tctcagccaa tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca 1740acttcttcgc ccccgttttc accatgcatg ggcaaatatt atacgcaagg cgacaaggtg 1800ctgatgccgc tggcgattca ggttcatcat gccgtctgtg atggcttcca tgtcggcaga 1860atgcttaatg aattacaaca gtactgcgat gagtggcagg gcggggcgta atttttttaa 1920ggcagttatt ggtgccctta aacgcctggg gtaatgactc tctagcttga ggcatcaaat 1980aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa 2040cgctctcctg agtaggacaa atccgccgct ctagagctgc ctcgcgcgtt tcggtgatga 2100cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga 2160tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc 2220agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca 2280gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg 2340agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 2400tgtcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 2460tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 2520aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 2580aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 2640ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 2700tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc 2760agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 2820gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 2880tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 2940acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 3000tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 3060caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 3120aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 3180aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 3240ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 3300agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 3360atagctgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 3420cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 3480aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 3540cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 3600aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 3660ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 3720gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 3780ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 3840tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 3900tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 3960ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 4020tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 4080agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 4140acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 4200ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 4260gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg 4320acattaacct ataaaaatag gcgtatcacg aggccctttc gtcttcac 4368 11 5064 DNAArtificial sequence pNCO-EC-MBP-BS-LuSy Expression vector 11 ctcgagaaatcataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca 60 attgtgagcggataacaatt tcacacagaa ttgattaaag aggagaaatt aactatgaaa 120 atcgaagaaggtaaactggt aatctggatt aacggcgata aaggctataa cggtctcgct 180 gaagtcggtaagaaattcga gaaagatacc ggaattaaag tcaccgttga gcatccggat 240 aaactggaagagaaattccc acaggttgcg gcaactggcg atggccctga cattatcttc 300 tgggcacacgaccgctttgg tggctacgct caatctggcc tgttggctga aatcaccccg 360 gacaaagcgttccaggacaa gctgtatccg tttacctggg atgccgtacg ttacaacggc 420 aagctgattgcttacccgat cgctgttgaa gcgttatcgc tgatttataa caaagatctg 480 ctgccgaacccgccaaaaac ctgggaagag atcccggcgc tggataaaga actgaaagcg 540 aaaggtaagagcgcgctgat gttcaacctg caagaaccgt acttcacctg gccgctgatt 600 gctgctgacgggggttatgc gttcaagtat gaaaacggca agtacgacat taaagacgtg 660 ggcgtggataacgctggcgc gaaagcgggt ctgaccttcc tggttgacct gattaaaaac 720 aaacacatgaatgcagacac cgattactcc atcgcagaag ctgcctttaa taaaggcgaa 780 acagcgatgaccatcaacgg cccgtgggca tggtccaaca tcgacaccag caaagtgaat 840 tatggtgtaacggtactgcc gaccttcaag ggtcaaccat ccaaaccgtt cgttggcgtg 900 ctgagcgcaggtattaacgc cgccagtccg aacaaagagc tggcaaaaga gttcctcgaa 960 aactatctgctgactgatga aggtctggaa gcggttaata aagacaaacc gctgggtgcc 1020 gtagcgctgaagtcttacga ggaagagttg gcgaaagatc cacgtattgc cgccaccatg 1080 gaaaacgcccagaaaggtga aatcatgccg aacatcccgc agatgtccgc tttctggtat 1140 gccgtgcgtactgcggtgat caacgccgcc agcggtcgtc agactgtcga tgaagccctg 1200 aaagacgcgcagactaattc gagctcgaac aacaacaaca ataacaataa caacaacctc 1260 gggatcgagggaaggatttc agaattcgcg gccgctatga atatcataca aggaaattta 1320 gttggtacaggtcttaaaat cggaatcgta gtaggaagat ttaatgattt tattacgagc 1380 aagctgctgagcggagcaga agatgcgctg ctcagacatg gcgtagacac aaatgacatt 1440 gatgtggcttgggttccagg cgcatttgaa ataccgtttg ctgcgaaaaa aatggcggaa 1500 acaaaaaaatatgatgctat tatcacattg ggcactgtca tcagaggcgc aacgacacat 1560 tacgattatgtctgcaatga agctgcaaaa ggcatcgcgc aagcagcaaa cactactggt 1620 gtacctgtcatctttggaat tgtaacaact gaaaacatcg aacaggctat cgagcgtgcc 1680 ggcacaaaagcgggcaacaa aggtgtagat tgtgctgttt ctgccattga aatggcaaat 1740 ttaaaccgctcatttgaata atttggatcc gtcgacctgc agccaagctt aattagctga 1800 gcttggactcctgttgatag atccagtaat gacctcagaa ctccatctgg atttgttcag 1860 aacgctcggttgccgccggg cgttttttat tggtgagaat ccaagctagc ttggcgagat 1920 tttcaggagctaaggaagct aaaatggaga aaaaaatcac tggatatacc accgttgata 1980 tatcccaatggcatcgtaaa gaacattttg aggcatttca gtcagttgct caatgtacct 2040 ataaccagaccgttcagctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc 2100 acaagttttatccggccttt attcacattc ttgcccgcct gatgaatgct catccggaat 2160 ttcgtatggcaatgaaagac ggtgagctgg tgatatggga tagtgttcac ccttgttaca 2220 ccgttttccatgagcaaact gaaacgtttt catcgctctg gagtgaatac cacgacgatt 2280 tccggcagtttctacacata tattcgcaag atgtggcgtg ttacggtgaa aacctggcct 2340 atttccctaaagggtttatt gagaatatgt ttttcgtctc agccaatccc tgggtgagtt 2400 tcaccagttttgatttaaac gtggccaata tggacaactt cttcgccccc gttttcacca 2460 tgcatgggcaaatattatac gcaaggcgac aaggtgctga tgccgctggc gattcaggtt 2520 catcatgccgtctgtgatgg cttccatgtc ggcagaatgc ttaatgaatt acaacagtac 2580 tgcgatgagtggcagggcgg ggcgtaattt ttttaaggca gttattggtg cccttaaacg 2640 cctggggtaatgactctcta gcttgaggca tcaaataaaa cgaaaggctc agtcgaaaga 2700 ctgggcctttcgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc 2760 gccgctctagagctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca 2820 gctcccggagacggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca 2880 gggcgcgtcagcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga 2940 tagcggagtgtatactggct taactatgcg gcatcagagc agattgtact gagagtgcac 3000 catatgcggtgtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct 3060 tccgcttcctcgctcactga ctcgctgcgc tcggtctgtc ggctgcggcg agcggtatca 3120 gctcactcaaaggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 3180 atgtgagcaaaaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 3240 ttccataggctccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 3300 cgaaacccgacaggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 3360 tctcctgttccgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 3420 gtggcgctttctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 3480 aagctgggctgtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 3540 tatcgtcttgagtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 3600 aacaggattagcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 3660 aactacggctacactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc 3720 ttcggaaaaagagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 3780 ttttttgtttgcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 3840 atcttttctacggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 3900 atgagattatcaaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 3960 tcaatctaaagtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 4020 gcacctatctcagcgatctg tctatttcgt tcatccatag ctgcctgact ccccgtcgtg 4080 tagataactacgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 4140 gacccacgctcaccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 4200 cgcagaagtggtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 4260 gctagagtaagtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 4320 atcgtggtgtcacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 4380 aggcgagttacatgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 4440 atcgttgtcagaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 4500 aattctcttactgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 4560 aagtcattctgagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 4620 gataataccgcgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 4680 gggcgaaaactctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 4740 gcacccaactgatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 4800 ggaaggcaaaatgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 4860 ctcttcctttttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 4920 atatttgaatgtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 4980 gtgccacctgacgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt 5040 atcacgaggccctttcgtct tcac 5064 12 4380 DNA Artificial sequencepNCO-BS-LuSy-EC-DHFR Expression vector 12 ctcgagaaat cataaaaaatttatttgctt tgtgagcgga taacaattat aatagattca 60 attgtgagcg gataacaatttcacacagaa ttcattaaag aggagaaatt aactatgaat 120 atcatacaag gaaatttagttggtacaggt cttaaaatcg gaatcgtagt aggaagattt 180 aatgatttta ttacgagcaagctgctgagc ggagcagaag atgcgctgct cagacatggc 240 gtagacacaa atgacattgatgtggcttgg gttccaggcg catttgaaat accgtttgct 300 gcgaaaaaaa tggcggaaacaaaaaaatat gatgctatta tcacattggg cactgtcatc 360 agaggcgcaa cgacacattacgattatgtc tgcaatgaag ctgcaaaagg catcgcgcaa 420 gcagcaaaca ctactggtgtacctgtcatc tttggaattg taacaactga aaacatcgaa 480 caggctatcg agcgtgccggcacaaaagcg ggcaacaaag gtgtagattg tgctgtttct 540 gccattgaaa tggcaaatttaaaccgctca tttgaattag cggccgctgg tggaggcgga 600 atgatcagtc tgattgcggcgttagcggta gatcgcgtta tcggcatgga aaacgccatg 660 ccgtggaacc tgcctgccgatctcgcctgg tttaaacgca acaccttaaa taaacccgtg 720 attatgggcc gccatacctgggaatcaatc ggtcgtccgt tgccaggacg caaaaatatt 780 atcctcagca gtcaaccgggtacggacgat cgcgtaacgt gggtgaagtc ggtggatgaa 840 gccatcgcgg cgtgtggtgacgtaccagaa atcatggtga ttggcggcgg tcgcgtttat 900 gaacagttct tgccaaaagcgcaaaaactg tatctgacgc atatcgacgc agaagtggaa 960 ggcgacaccc atttcccggattacgagccg gatgactggg aatcggtatt cagcgaattc 1020 cacgatgctg atgcgcagaactctcacagc tattgctttg agattctgga gcggcggtaa 1080 ggatccgtcg acctgcagccaagcttaatt agctgagctt ggactcctgt tgatagatcc 1140 agtaatgacc tcagaactccatctggattt gttcagaacg ctcggttgcc gccgggcgtt 1200 ttttattggt gagaatccaagctagcttgg cgagattttc aggagctaag gaagctaaaa 1260 tggagaaaaa aatcactggatataccaccg ttgatatatc ccaatggcat cgtaaagaac 1320 attttgaggc atttcagtcagttgctcaat gtacctataa ccagaccgtt cagctggata 1380 ttacggcctt tttaaagaccgtaaagaaaa ataagcacaa gttttatccg gcctttattc 1440 acattcttgc ccgcctgatgaatgctcatc cggaatttcg tatggcaatg aaagacggtg 1500 agctggtgat atgggatagtgttcaccctt gttacaccgt tttccatgag caaactgaaa 1560 cgttttcatc gctctggagtgaataccacg acgatttccg gcagtttcta cacatatatt 1620 cgcaagatgt ggcgtgttacggtgaaaacc tggcctattt ccctaaaggg tttattgaga 1680 atatgttttt cgtctcagccaatccctggg tgagtttcac cagttttgat ttaaacgtgg 1740 ccaatatgga caacttcttcgcccccgttt tcaccatgca tgggcaaata ttatacgcaa 1800 ggcgacaagg tgctgatgccgctggcgatt caggttcatc atgccgtctg tgatggcttc 1860 catgtcggca gaatgcttaatgaattacaa cagtactgcg atgagtggca gggcggggcg 1920 taattttttt aaggcagttattggtgccct taaacgcctg gggtaatgac tctctagctt 1980 gaggcatcaa ataaaacgaaaggctcagtc gaaagactgg gcctttcgtt ttatctgttg 2040 tttgtcggtg aacgctctcctgagtaggac aaatccgccg ctctagagct gcctcgcgcg 2100 tttcggtgat gacggtgaaaacctctgaca catgcagctc ccggagacgg tcacagcttg 2160 tctgtaagcg gatgccgggagcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg 2220 gtgtcggggc gcagccatgacccagtcacg tagcgatagc ggagtgtata ctggcttaac 2280 tatgcggcat cagagcagattgtactgaga gtgcaccata tgcggtgtga aataccgcac 2340 agatgcgtaa ggagaaaataccgcatcagg cgctcttccg cttcctcgct cactgactcg 2400 ctgcgctcgg tctgtcggctgcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 2460 ttatccacag aatcaggggataacgcagga aagaacatgt gagcaaaagg ccagcaaaag 2520 gccaggaacc gtaaaaaggccgcgttgctg gcgtttttcc ataggctccg cccccctgac 2580 gagcatcaca aaaatcgacgctcaagtcag aggtggcgaa acccgacagg actataaaga 2640 taccaggcgt ttccccctggaagctccctc gtgcgctctc ctgttccgac cctgccgctt 2700 accggatacc tgtccgcctttctcccttcg ggaagcgtgg cgctttctca atgctcacgc 2760 tgtaggtatc tcagttcggtgtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 2820 cccgttcagc ccgaccgctgcgccttatcc ggtaactatc gtcttgagtc caacccggta 2880 agacacgact tatcgccactggcagcagcc actggtaaca ggattagcag agcgaggtat 2940 gtaggcggtg ctacagagttcttgaagtgg tggcctaact acggctacac tagaaggaca 3000 gtatttggta tctgcgctctgctgaagcca gttaccttcg gaaaaagagt tggtagctct 3060 tgatccggca aacaaaccaccgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 3120 acgcgcagaa aaaaaggatctcaagaagat cctttgatct tttctacggg gtctgacgct 3180 cagtggaacg aaaactcacgttaagggatt ttggtcatga gattatcaaa aaggatcttc 3240 acctagatcc ttttaaattaaaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 3300 acttggtctg acagttaccaatgcttaatc agtgaggcac ctatctcagc gatctgtcta 3360 tttcgttcat ccatagctgcctgactcccc gtcgtgtaga taactacgat acgggagggc 3420 ttaccatctg gccccagtgctgcaatgata ccgcgagacc cacgctcacc ggctccagat 3480 ttatcagcaa taaaccagccagccggaagg gccgagcgca gaagtggtcc tgcaacttta 3540 tccgcctcca tccagtctattaattgttgc cgggaagcta gagtaagtag ttcgccagtt 3600 aatagtttgc gcaacgttgttgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 3660 ggtatggctt cattcagctccggttcccaa cgatcaaggc gagttacatg atcccccatg 3720 ttgtgcaaaa aagcggttagctccttcggt cctccgatcg ttgtcagaag taagttggcc 3780 gcagtgttat cactcatggttatggcagca ctgcataatt ctcttactgt catgccatcc 3840 gtaagatgct tttctgtgactggtgagtac tcaaccaagt cattctgaga atagtgtatg 3900 cggcgaccga gttgctcttgcccggcgtca atacgggata ataccgcgcc acatagcaga 3960 actttaaaag tgctcatcattggaaaacgt tcttcggggc gaaaactctc aaggatctta 4020 ccgctgttga gatccagttcgatgtaaccc actcgtgcac ccaactgatc ttcagcatct 4080 tttactttca ccagcgtttctgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 4140 ggaataaggg cgacacggaaatgttgaata ctcatactct tcctttttca atattattga 4200 agcatttatc agggttattgtctcatgagc ggatacatat ttgaatgtat ttagaaaaat 4260 aaacaaatag gggttccgcgcacatttccc cgaaaagtgc cacctgacgt ctaagaaacc 4320 attattatca tgacattaacctataaaaat aggcgtatca cgaggccctt tcgtcttcac 4380 13 3936 DNA Artificialsequence pNCO-N-VP2-BS-LuSy Expression vector 13 ctcgagaaat cataaaaaatttatttgctt tgtgagcgga taacaattat aatagattca 60 attgtgagcg gataacaatttcacacagaa ttcattaaag aggagaaatt aactatgggg 120 gacggtgctg ttcagccggacggtggtcag ccggctgttc gtaacgaacg tatgaatatc 180 atacaaggaa atttagttggtacaggtctt aaaatcggaa tcgtagtagg aagatttaat 240 gattttatta cgagcaagctgctgagcgga gcagaagatg cgctgctcag acatggcgta 300 gacacaaatg acattgatgtggcttgggtt ccaggcgcat ttgaaatacc gtttgctgcg 360 aaaaaaatgg cggaaacaaaaaaatatgat gctattatca cattgggcac tgtcatcaga 420 ggcgcaacga cacattacgattatgtctgc aatgaagctg caaaaggcat cgcgcaagca 480 gcaaacacta ctggtgtacctgtcatcttt ggaattgtaa caactgaaaa catcgaacag 540 gctatcgagc gtgccggcacaaaagcgggc aacaaaggtg tagattgtgc tgtttctgcc 600 attgaaatgg caaatttaaaccgctcattt gaataaggat ccgtcgacct gcagccaagc 660 ttaattagct gagcttggactcctgttgat agatccagta atgacctcag aactccatct 720 ggatttgttc agaacgctcggttgccgccg ggcgtttttt attggtgaga atccaagcta 780 gcttggcgag attttcaggagctaaggaag ctaaaatgga gaaaaaaatc actggatata 840 ccaccgttga tatatcccaatggcatcgta aagaacattt tgaggcattt cagtcagttg 900 ctcaatgtac ctataaccagaccgttcagc tggatattac ggccttttta aagaccgtaa 960 agaaaaataa gcacaagttttatccggcct ttattcacat tcttgcccgc ctgatgaatg 1020 ctcatccgga atttcgtatggcaatgaaag acggtgagct ggtgatatgg gatagtgttc 1080 acccttgtta caccgttttccatgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat 1140 accacgacga tttccggcagtttctacaca tatattcgca agatgtggcg tgttacggtg 1200 aaaacctggc ctatttccctaaagggttta ttgagaatat gtttttcgtc tcagccaatc 1260 cctgggtgag tttcaccagttttgatttaa acgtggccaa tatggacaac ttcttcgccc 1320 ccgttttcac catgcatgggcaaatattat acgcaaggcg acaaggtgct gatgccgctg 1380 gcgattcagg ttcatcatgccgtctgtgat ggcttccatg tcggcagaat gcttaatgaa 1440 ttacaacagt actgcgatgagtggcagggc ggggcgtaat ttttttaagg cagttattgg 1500 tgcccttaaa cgcctggggtaatgactctc tagcttgagg catcaaataa aacgaaaggc 1560 tcagtcgaaa gactgggcctttcgttttat ctgttgtttg tcggtgaacg ctctcctgag 1620 taggacaaat ccgccgctctagagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct 1680 ctgacacatg cagctcccggagacggtcac agcttgtctg taagcggatg ccgggagcag 1740 acaagcccgt cagggcgcgtcagcgggtgt tggcgggtgt cggggcgcag ccatgaccca 1800 gtcacgtagc gatagcggagtgtatactgg cttaactatg cggcatcaga gcagattgta 1860 ctgagagtgc accatatgcggtgtgaaata ccgcacagat gcgtaaggag aaaataccgc 1920 atcaggcgct cttccgcttcctcgctcact gactcgctgc gctcggtctg tcggctgcgg 1980 cgagcggtat cagctcactcaaaggcggta atacggttat ccacagaatc aggggataac 2040 gcaggaaaga acatgtgagcaaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 2100 ttgctggcgt ttttccataggctccgcccc cctgacgagc atcacaaaaa tcgacgctca 2160 agtcagaggt ggcgaaacccgacaggacta taaagatacc aggcgtttcc ccctggaagc 2220 tccctcgtgc gctctcctgttccgaccctg ccgcttaccg gatacctgtc cgcctttctc 2280 ccttcgggaa gcgtggcgctttctcaatgc tcacgctgta ggtatctcag ttcggtgtag 2340 gtcgttcgct ccaagctgggctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 2400 ttatccggta actatcgtcttgagtccaac ccggtaagac acgacttatc gccactggca 2460 gcagccactg gtaacaggattagcagagcg aggtatgtag gcggtgctac agagttcttg 2520 aagtggtggc ctaactacggctacactaga aggacagtat ttggtatctg cgctctgctg 2580 aagccagtta ccttcggaaaaagagttggt agctcttgat ccggcaaaca aaccaccgct 2640 ggtagcggtg gtttttttgtttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 2700 gaagatcctt tgatcttttctacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 2760 gggattttgg tcatgagattatcaaaaagg atcttcacct agatcctttt aaattaaaaa 2820 tgaagtttta aatcaatctaaagtatatat gagtaaactt ggtctgacag ttaccaatgc 2880 ttaatcagtg aggcacctatctcagcgatc tgtctatttc gttcatccat agctgcctga 2940 ctccccgtcg tgtagataactacgatacgg gagggcttac catctggccc cagtgctgca 3000 atgataccgc gagacccacgctcaccggct ccagatttat cagcaataaa ccagccagcc 3060 ggaagggccg agcgcagaagtggtcctgca actttatccg cctccatcca gtctattaat 3120 tgttgccggg aagctagagtaagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 3180 attgctacag gcatcgtggtgtcacgctcg tcgtttggta tggcttcatt cagctccggt 3240 tcccaacgat caaggcgagttacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 3300 ttcggtcctc cgatcgttgtcagaagtaag ttggccgcag tgttatcact catggttatg 3360 gcagcactgc ataattctcttactgtcatg ccatccgtaa gatgcttttc tgtgactggt 3420 gagtactcaa ccaagtcattctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 3480 gcgtcaatac gggataataccgcgccacat agcagaactt taaaagtgct catcattgga 3540 aaacgttctt cggggcgaaaactctcaagg atcttaccgc tgttgagatc cagttcgatg 3600 taacccactc gtgcacccaactgatcttca gcatctttta ctttcaccag cgtttctggg 3660 tgagcaaaaa caggaaggcaaaatgccgca aaaaagggaa taagggcgac acggaaatgt 3720 tgaatactca tactcttcctttttcaatat tattgaagca tttatcaggg ttattgtctc 3780 atgagcggat acatatttgaatgtatttag aaaaataaac aaataggggt tccgcgcaca 3840 tttccccgaa aagtgccacctgacgtctaa gaaaccatta ttatcatgac attaacctat 3900 aaaaataggc gtatcacgaggccctttcgt cttcac 3936 14 3932 DNA Artificial sequencepNCO-C-VP2-BS-LuSy Expression vector 14 ctcgagaaat cataaaaaat ttatttgctttgtgagcgga taacaattat aatagattca 60 attgtgagcg gataacaatt tcacacagaattcattaaag aggagaaatt aactatgaat 120 atcatacaag gaaatttagt tggtacaggtcttaaaatcg gaatcgtagt aggaagattt 180 aatgatttta ttacgagcaa gctgctgagcggagcagaag atgcgctgct cagacatggc 240 gtagacacaa atgacattga tgtggcttgggttccaggcg catttgaaat accgtttgct 300 gcgaaaaaaa tggcggaaac aaaaaaatatgatgctatta tcacattggg cactgtcatc 360 agaggcgcaa cgacacatta cgattatgtctgcaatgaag ctgcaaaagg catcgcgcaa 420 gcagcaaaca ctactggtgt acctgtcatctttggaattg taacaactga aaacatcgaa 480 caggctatcg agcgtgccgg cacaaaagcgggcaacaaag gtgtagattg tgctgtttct 540 gccattgaaa tggcaaactt aaaccgttctttcgaaggtg acggtgctgt tcagccggac 600 ggtggtcagc cggctgttcg taacgaacgttaggatccgt cgacctgcag ccaagcttaa 660 ttagctgagc ttggactcct gttgatagatccagtaatga cctcagaact ccatctggat 720 ttgttcagaa cgctcggttg ccgccgggcgttttttattg gtgagaatcc aagctagctt 780 ggcgagattt tcaggagcta aggaagctaaaatggagaaa aaaatcactg gatataccac 840 cgttgatata tcccaatggc atcgtaaagaacattttgag gcatttcagt cagttgctca 900 atgtacctat aaccagaccg ttcagctggatattacggcc tttttaaaga ccgtaaagaa 960 aaataagcac aagttttatc cggcctttattcacattctt gcccgcctga tgaatgctca 1020 tccggaattt cgtatggcaa tgaaagacggtgagctggtg atatgggata gtgttcaccc 1080 ttgttacacc gttttccatg agcaaactgaaacgttttca tcgctctgga gtgaatacca 1140 cgacgatttc cggcagtttc tacacatatattcgcaagat gtggcgtgtt acggtgaaaa 1200 cctggcctat ttccctaaag ggtttattgagaatatgttt ttcgtctcag ccaatccctg 1260 ggtgagtttc accagttttg atttaaacgtggccaatatg gacaacttct tcgcccccgt 1320 tttcaccatg catgggcaaa tattatacgcaaggcgacaa ggtgctgatg ccgctggcga 1380 ttcaggttca tcatgccgtc tgtgatggcttccatgtcgg cagaatgctt aatgaattac 1440 aacagtactg cgatgagtgg cagggcggggcgtaattttt ttaaggcagt tattggtgcc 1500 cttaaacgcc tggggtaatg actctctagcttgaggcatc aaataaaacg aaaggctcag 1560 tcgaaagact gggcctttcg ttttatctgttgtttgtcgg tgaacgctct cctgagtagg 1620 acaaatccgc cgctctagag ctgcctcgcgcgtttcggtg atgacggtga aaacctctga 1680 cacatgcagc tcccggagac ggtcacagcttgtctgtaag cggatgccgg gagcagacaa 1740 gcccgtcagg gcgcgtcagc gggtgttggcgggtgtcggg gcgcagccat gacccagtca 1800 cgtagcgata gcggagtgta tactggcttaactatgcggc atcagagcag attgtactga 1860 gagtgcacca tatgcggtgt gaaataccgcacagatgcgt aaggagaaaa taccgcatca 1920 ggcgctcttc cgcttcctcg ctcactgactcgctgcgctc ggtctgtcgg ctgcggcgag 1980 cggtatcagc tcactcaaag gcggtaatacggttatccac agaatcaggg gataacgcag 2040 gaaagaacat gtgagcaaaa ggccagcaaaaggccaggaa ccgtaaaaag gccgcgttgc 2100 tggcgttttt ccataggctc cgcccccctgacgagcatca caaaaatcga cgctcaagtc 2160 agaggtggcg aaacccgaca ggactataaagataccaggc gtttccccct ggaagctccc 2220 tcgtgcgctc tcctgttccg accctgccgcttaccggata cctgtccgcc tttctccctt 2280 cgggaagcgt ggcgctttct caatgctcacgctgtaggta tctcagttcg gtgtaggtcg 2340 ttcgctccaa gctgggctgt gtgcacgaaccccccgttca gcccgaccgc tgcgccttat 2400 ccggtaacta tcgtcttgag tccaacccggtaagacacga cttatcgcca ctggcagcag 2460 ccactggtaa caggattagc agagcgaggtatgtaggcgg tgctacagag ttcttgaagt 2520 ggtggcctaa ctacggctac actagaaggacagtatttgg tatctgcgct ctgctgaagc 2580 cagttacctt cggaaaaaga gttggtagctcttgatccgg caaacaaacc accgctggta 2640 gcggtggttt ttttgtttgc aagcagcagattacgcgcag aaaaaaagga tctcaagaag 2700 atcctttgat cttttctacg gggtctgacgctcagtggaa cgaaaactca cgttaaggga 2760 ttttggtcat gagattatca aaaaggatcttcacctagat ccttttaaat taaaaatgaa 2820 gttttaaatc aatctaaagt atatatgagtaaacttggtc tgacagttac caatgcttaa 2880 tcagtgaggc acctatctca gcgatctgtctatttcgttc atccatagct gcctgactcc 2940 ccgtcgtgta gataactacg atacgggagggcttaccatc tggccccagt gctgcaatga 3000 taccgcgaga cccacgctca ccggctccagatttatcagc aataaaccag ccagccggaa 3060 gggccgagcg cagaagtggt cctgcaactttatccgcctc catccagtct attaattgtt 3120 gccgggaagc tagagtaagt agttcgccagttaatagttt gcgcaacgtt gttgccattg 3180 ctacaggcat cgtggtgtca cgctcgtcgtttggtatggc ttcattcagc tccggttccc 3240 aacgatcaag gcgagttaca tgatcccccatgttgtgcaa aaaagcggtt agctccttcg 3300 gtcctccgat cgttgtcaga agtaagttggccgcagtgtt atcactcatg gttatggcag 3360 cactgcataa ttctcttact gtcatgccatccgtaagatg cttttctgtg actggtgagt 3420 actcaaccaa gtcattctga gaatagtgtatgcggcgacc gagttgctct tgcccggcgt 3480 caatacggga taataccgcg ccacatagcagaactttaaa agtgctcatc attggaaaac 3540 gttcttcggg gcgaaaactc tcaaggatcttaccgctgtt gagatccagt tcgatgtaac 3600 ccactcgtgc acccaactga tcttcagcatcttttacttt caccagcgtt tctgggtgag 3660 caaaaacagg aaggcaaaat gccgcaaaaaagggaataag ggcgacacgg aaatgttgaa 3720 tactcatact cttccttttt caatattattgaagcattta tcagggttat tgtctcatga 3780 gcggatacat atttgaatgt atttagaaaaataaacaaat aggggttccg cgcacatttc 3840 cccgaaaagt gccacctgac gtctaagaaaccattattat catgacatta acctataaaa 3900 ataggcgtat cacgaggccc tttcgtcttcac 3932 15 3989 DNA Artificial sequence pNCO-N/C-VP2-BS-LuSy Expressionvector 15 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattataatagattca 60 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaattaactatgggg 120 gacggtgctg ttcagccgga cggtggtcag ccggctgttc gtaacgaacgtatgaatatc 180 atacaaggaa atttagttgg tacaggtctt aaaatcggaa tcgtagtaggaagatttaat 240 gattttatta cgagcaagct gctgagcgga gcagaagatg cgctgctcagacatggcgta 300 gacacaaatg acattgatgt ggcttgggtt ccaggcgcat ttgaaataccgtttgctgcg 360 aaaaaaatgg cggaaacaaa aaaatatgat gctattatca cattgggcactgtcatcaga 420 ggcgcaacga cacattacga ttatgtctgc aatgaagctg caaaaggcatcgcgcaagca 480 gcaaacacta ctggtgtacc tgtcatcttt ggaattgtaa caactgaaaacatcgaacag 540 gctatcgagc gtgccggcac aaaagcgggc aacaaaggtg tagattgtgctgtttctgcc 600 attgaaatgg caaacttaaa ccgttctttc gaaggtgacg gtgctgttcagccggacggt 660 ggtcagccgg ctgttcgtaa cgaacgttag gatccgtcga cctgcagccaagcttaatta 720 gctgagcttg gactcctgtt gatagatcca gtaatgacct cagaactccatctggatttg 780 ttcagaacgc tcggttgccg ccgggcgttt tttattggtg agaatccaagctagcttggc 840 gagattttca ggagctaagg aagctaaaat ggagaaaaaa atcactggatataccaccgt 900 tgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcagttgctcaatg 960 tacctataac cagaccgttc agctggatat tacggccttt ttaaagaccgtaaagaaaaa 1020 taagcacaag ttttatccgg cctttattca cattcttgcc cgcctgatgaatgctcatcc 1080 ggaatttcgt atggcaatga aagacggtga gctggtgata tgggatagtgttcacccttg 1140 ttacaccgtt ttccatgagc aaactgaaac gttttcatcg ctctggagtgaataccacga 1200 cgatttccgg cagtttctac acatatattc gcaagatgtg gcgtgttacggtgaaaacct 1260 ggcctatttc cctaaagggt ttattgagaa tatgtttttc gtctcagccaatccctgggt 1320 gagtttcacc agttttgatt taaacgtggc caatatggac aacttcttcgcccccgtttt 1380 caccatgcat gggcaaatat tatacgcaag gcgacaaggt gctgatgccgctggcgattc 1440 aggttcatca tgccgtctgt gatggcttcc atgtcggcag aatgcttaatgaattacaac 1500 agtactgcga tgagtggcag ggcggggcgt aattttttta aggcagttattggtgccctt 1560 aaacgcctgg ggtaatgact ctctagcttg aggcatcaaa taaaacgaaaggctcagtcg 1620 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcctgagtaggaca 1680 aatccgccgc tctagagctg cctcgcgcgt ttcggtgatg acggtgaaaacctctgacac 1740 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggagcagacaagcc 1800 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgacccagtcacgt 1860 agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagattgtactgagag 1920 tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaataccgcatcaggc 1980 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt ctgtcggctgcggcgagcgg 2040 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggataacgcaggaa 2100 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggccgcgttgctgg 2160 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgctcaagtcaga 2220 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctggaagctccctcg 2280 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgcctttctcccttcgg 2340 gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtgtaggtcgttc 2400 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgcgccttatccg 2460 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactggcagcagcca 2520 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttcttgaagtggt 2580 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctgctgaagccag 2640 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccaccgctggtagcg 2700 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatctcaagaagatc 2760 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgttaagggattt 2820 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaaaaatgaagtt 2880 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaatgcttaatca 2940 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagctgcctgactccccg 3000 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgctgcaatgatac 3060 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagccagccggaaggg 3120 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctattaattgttgcc 3180 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgttgccattgcta 3240 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctccggttcccaac 3300 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagctccttcggtc 3360 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggttatggcagcac 3420 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgactggtgagtact 3480 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgcccggcgtcaa 3540 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcattggaaaacgtt 3600 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcgatgtaaccca 3660 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttctgggtgagcaa 3720 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaatgttgaatac 3780 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgtctcatgagcg 3840 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgcacatttcccc 3900 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacctataaaaata 3960 ggcgtatcac gaggcccttt cgtcttcac 3989 16 3927 DNAArtificial sequence pNCO-C-Biotag-BS-LuSy Expression vector 16ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca 60attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaat 120atcatacaag gaaatttagt tggtacaggt cttaaaatcg gaatcgtagt aggaagattt 180aatgatttta ttacgagcaa gctgctgagc ggagcagaag atgcgctgct cagacatggc 240gtagacacaa atgacattga tgtggcttgg gttccaggcg catttgaaat accgtttgct 300gcgaaaaaaa tggcggaaac aaaaaaatat gatgctatta tcacattggg cactgtcatc 360agaggcgcaa cgacacatta cgattatgtc tgcaatgaag ctgcaaaagg catcgcgcaa 420gcagcaaaca ctactggtgt acctgtcatc tttggaattg taacaactga aaacatcgaa 480caggctatcg agcgtgccgg cacaaaagcg ggcaacaaag gtgtagattg tgctgtttct 540gccattgaaa tggcaaactt aaaccgttct ttcgaagcgg ccgcactcgg cggcatcttc 600gaagctatga agatggagtg gcgctaagga tccgtcgacc tgcagccaag cttaattagc 660tgagcttgga ctcctgttga tagatccagt aatgacctca gaactccatc tggatttgtt 720cagaacgctc ggttgccgcc gggcgttttt tattggtgag aatccaagct agcttggcga 780gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg 840atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta 900cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaaata 960agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg 1020aatttcgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt 1080acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg 1140atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg 1200cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga 1260gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca 1320ccatgcatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag 1380gttcatcatg ccgtctgtga tggcttccat gtcggcagaa tgcttaatga attacaacag 1440tactgcgatg agtggcaggg cggggcgtaa tttttttaag gcagttattg gtgcccttaa 1500acgcctgggg taatgactct ctagcttgag gcatcaaata aaacgaaagg ctcagtcgaa 1560agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa 1620tccgccgctc tagagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat 1680gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg 1740tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag 1800cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg 1860caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc 1920tcttccgctt cctcgctcac tgactcgctg cgctcggtct gtcggctgcg gcgagcggta 1980tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 2040aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 2100tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 2160tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 2220cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 2280agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 2340tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 2400aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 2460ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 2520cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt 2580accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 2640ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 2700ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 2760gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 2820aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 2880gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagctgcctg actccccgtc 2940gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 3000cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 3060gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 3120gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 3180ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 3240tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 3300ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 3360cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 3420accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 3480cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 3540tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 3600cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 3660acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 3720atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 3780tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 3840aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg 3900cgtatcacga ggccctttcg tcttcac 3927 17 3912 DNA Artificial sequencepNCO-Lys165-BS-LuSy Expression vector 17 ctcgagaaat cataaaaaatttatttgctt tgtgagcgga taacaattat aatagattca 60 attgtgagcg gataacaatttcacacagaa ttcattaaag aggagaaatt aactatgaat 120 atcatacaag gaaatttagttggtacaggt cttaaaatcg gaatcgtagt aggaagattt 180 aatgatttta ttacgagcaagctgctgagc ggagcagaag atgcgctgct cagacatggc 240 gtagacacaa atgacattgatgtggcttgg gttccaggcg catttgaaat accgtttgct 300 gcgaaaaaaa tggcggaaacaaaaaaatat gatgctatta tcacattggg cactgtcatc 360 agaggcgcaa cgacacattacgattatgtc tgcaatgaag ctgcaaaagg catcgcgcaa 420 gcagcaaaca ctactggtgtacctgtcatc tttggaattg taacaactga aaacatcgaa 480 caggctatcg agcgtgccggcacaaaagcg ggcaacaaag gtgtagattg tgctgtttct 540 gccattgaaa tggcaaacttaaaccgttct ttcgaaggtg gcggtggttc tggtggtggc 600 tctggtaaat aaggatccgtcgacctgcag ccaagcttaa ttagctgagc ttggactcct 660 gttgatagat ccagtaatgacctcagaact ccatctggat ttgttcagaa cgctcggttg 720 ccgccgggcg ttttttattggtgagaatcc aagctagctt ggcgagattt tcaggagcta 780 aggaagctaa aatggagaaaaaaatcactg gatataccac cgttgatata tcccaatggc 840 atcgtaaaga acattttgaggcatttcagt cagttgctca atgtacctat aaccagaccg 900 ttcagctgga tattacggcctttttaaaga ccgtaaagaa aaataagcac aagttttatc 960 cggcctttat tcacattcttgcccgcctga tgaatgctca tccggaattt cgtatggcaa 1020 tgaaagacgg tgagctggtgatatgggata gtgttcaccc ttgttacacc gttttccatg 1080 agcaaactga aacgttttcatcgctctgga gtgaatacca cgacgatttc cggcagtttc 1140 tacacatata ttcgcaagatgtggcgtgtt acggtgaaaa cctggcctat ttccctaaag 1200 ggtttattga gaatatgtttttcgtctcag ccaatccctg ggtgagtttc accagttttg 1260 atttaaacgt ggccaatatggacaacttct tcgcccccgt tttcaccatg catgggcaaa 1320 tattatacgc aaggcgacaaggtgctgatg ccgctggcga ttcaggttca tcatgccgtc 1380 tgtgatggct tccatgtcggcagaatgctt aatgaattac aacagtactg cgatgagtgg 1440 cagggcgggg cgtaatttttttaaggcagt tattggtgcc cttaaacgcc tggggtaatg 1500 actctctagc ttgaggcatcaaataaaacg aaaggctcag tcgaaagact gggcctttcg 1560 ttttatctgt tgtttgtcggtgaacgctct cctgagtagg acaaatccgc cgctctagag 1620 ctgcctcgcg cgtttcggtgatgacggtga aaacctctga cacatgcagc tcccggagac 1680 ggtcacagct tgtctgtaagcggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc 1740 gggtgttggc gggtgtcggggcgcagccat gacccagtca cgtagcgata gcggagtgta 1800 tactggctta actatgcggcatcagagcag attgtactga gagtgcacca tatgcggtgt 1860 gaaataccgc acagatgcgtaaggagaaaa taccgcatca ggcgctcttc cgcttcctcg 1920 ctcactgact cgctgcgctcggtctgtcgg ctgcggcgag cggtatcagc tcactcaaag 1980 gcggtaatac ggttatccacagaatcaggg gataacgcag gaaagaacat gtgagcaaaa 2040 ggccagcaaa aggccaggaaccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 2100 cgcccccctg acgagcatcacaaaaatcga cgctcaagtc agaggtggcg aaacccgaca 2160 ggactataaa gataccaggcgtttccccct ggaagctccc tcgtgcgctc tcctgttccg 2220 accctgccgc ttaccggatacctgtccgcc tttctccctt cgggaagcgt ggcgctttct 2280 caatgctcac gctgtaggtatctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 2340 gtgcacgaac cccccgttcagcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 2400 tccaacccgg taagacacgacttatcgcca ctggcagcag ccactggtaa caggattagc 2460 agagcgaggt atgtaggcggtgctacagag ttcttgaagt ggtggcctaa ctacggctac 2520 actagaagga cagtatttggtatctgcgct ctgctgaagc cagttacctt cggaaaaaga 2580 gttggtagct cttgatccggcaaacaaacc accgctggta gcggtggttt ttttgtttgc 2640 aagcagcaga ttacgcgcagaaaaaaagga tctcaagaag atcctttgat cttttctacg 2700 gggtctgacg ctcagtggaacgaaaactca cgttaaggga ttttggtcat gagattatca 2760 aaaaggatct tcacctagatccttttaaat taaaaatgaa gttttaaatc aatctaaagt 2820 atatatgagt aaacttggtctgacagttac caatgcttaa tcagtgaggc acctatctca 2880 gcgatctgtc tatttcgttcatccatagct gcctgactcc ccgtcgtgta gataactacg 2940 atacgggagg gcttaccatctggccccagt gctgcaatga taccgcgaga cccacgctca 3000 ccggctccag atttatcagcaataaaccag ccagccggaa gggccgagcg cagaagtggt 3060 cctgcaactt tatccgcctccatccagtct attaattgtt gccgggaagc tagagtaagt 3120 agttcgccag ttaatagtttgcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 3180 cgctcgtcgt ttggtatggcttcattcagc tccggttccc aacgatcaag gcgagttaca 3240 tgatccccca tgttgtgcaaaaaagcggtt agctccttcg gtcctccgat cgttgtcaga 3300 agtaagttgg ccgcagtgttatcactcatg gttatggcag cactgcataa ttctcttact 3360 gtcatgccat ccgtaagatgcttttctgtg actggtgagt actcaaccaa gtcattctga 3420 gaatagtgta tgcggcgaccgagttgctct tgcccggcgt caatacggga taataccgcg 3480 ccacatagca gaactttaaaagtgctcatc attggaaaac gttcttcggg gcgaaaactc 3540 tcaaggatct taccgctgttgagatccagt tcgatgtaac ccactcgtgc acccaactga 3600 tcttcagcat cttttactttcaccagcgtt tctgggtgag caaaaacagg aaggcaaaat 3660 gccgcaaaaa agggaataagggcgacacgg aaatgttgaa tactcatact cttccttttt 3720 caatattatt gaagcatttatcagggttat tgtctcatga gcggatacat atttgaatgt 3780 atttagaaaa ataaacaaataggggttccg cgcacatttc cccgaaaagt gccacctgac 3840 gtctaagaaa ccattattatcatgacatta acctataaaa ataggcgtat cacgaggccc 3900 tttcgtcttc ac 3912 183919 DNA Artificial sequence pNCO-Cys167-LuSy Expression vector 18ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca 60attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaat 120atcatacaag gaaatttagt tggtacaggt cttaaaatcg gaatcgtagt aggaagattt 180aatgatttta ttacgagcaa gctgctgagc ggagcagaag atgcgctgct cagacatggc 240gtagacacaa atgacattga tgtggcttgg gttccaggcg catttgaaat accgtttgct 300gcgaaaaaaa tggcggaaac aaaaaaatat gatgctatta tcacattggg cactgtcatc 360agaggcgcaa cgacacatta cgattatgtc tgcaatgaag ctgcaaaagg catcgcgcaa 420gcagcaaaca ctactggtgt acctgtcatc tttggaattg taacaactga aaacatcgaa 480caggctatcg agcgtgccgg cacaaaagcg ggcaacaaag gtgtagattg tgctgtttct 540gccattgaaa tggcaaactt aaaccgttct ttcgaaggtg gcggtggttc tggtggtggc 600tctggtggtg gctgctaagg atcccgtcga cctgcagcca agcttaatta gctgagcttg 660gactcctgtt gatagatcca gtaatgacct cagaactcca tctggatttg ttcagaacgc 720tcggttgccg ccgggcgttt tttattggtg agaatccaag ctagcttggc gagattttca 780ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc 840caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac 900cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag 960ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc ggaatttcgt 1020atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt 1080ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg 1140cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc 1200cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc 1260agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgcat 1320gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca 1380tgccgtctgt gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga 1440tgagtggcag ggcggggcgt aattttttta aggcagttat tggtgccctt aaacgcctgg 1500ggtaatgact ctctagcttg aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg 1560cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgc 1620tctagagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc 1680cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg 1740cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg 1800gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat 1860gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc 1920ttcctcgctc actgactcgc tgcgctcggt ctgtcggctg cggcgagcgg tatcagctca 1980ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 2040agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 2100taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 2160cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 2220tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 2280gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 2340gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 2400tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 2460gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 2520cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 2580aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 2640tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 2700ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 2760attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 2820ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 2880tatctcagcg atctgtctat ttcgttcatc catagctgcc tgactccccg tcgtgtagat 2940aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 3000acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 3060aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 3120agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 3180ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 3240agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 3300tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 3360tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 3420attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 3480taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 3540aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 3600caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 3660gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 3720cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 3780tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 3840acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac 3900gaggcccttt cgtcttcac 3919 19 5531 DNA Artificial sequencepFLAG-MAC-BS-LuSy Expression vector 19 catcataacg gttctggcaa atattctgaaatgagctgtt gacaattaat catcggctcg 60 tataatgtgt ggaattgtga gcggataacaatttcacaca ggagatatct tatggactac 120 aaggacgacg atgacaaagt caagcttatgaatatcatac aaggaaattt agttggtaca 180 ggtcttaaaa tcggaatcgt agtaggaagatttaatgatt ttattacgag caagctgctg 240 agcggagcag aagatgcgct gctcagacatggcgtagaca caaatgacat tgatgtggct 300 tgggttccag gcgcatttga aataccgtttgctgcgaaaa aaatggcgga aacaaaaaaa 360 tatgatgcta ttatcacatt gggcactgtcatcagaggcg caacgacaca ttacgattat 420 gtctgcaatg aagctgcaaa aggcatcgcgcaagcagcaa acactactgg tgtacctgtc 480 atctttggaa ttgtaacaac tgaaaacatcgaacaggcta tcgagcgtgc cggcacaaaa 540 gcgggcaaca aaggtgtaga ttgtgctgtttctgccattg aaatggcaaa cttaaaccgt 600 tctttcgaat aagaattccc gggtacctgcagatctagat agatgagctc gtcgagtgag 660 agaagatttt cagcctgata cagattaaatcagaagcggt ctgataaaac agaatttgcc 720 tggcggcagt agcgcggtgg tcccacctgaccccatgccg aactcagaag tgaaacgccg 780 tagcgccgat ggtagtgtgg ggtctccccatgcgagagta gggaactgcc aggcatcaaa 840 taaaacgaaa ggctcagtcg aaagactgggcctttcgttt tatctgttgt ttgtcggtga 900 acgctctcct gagtaggaca aatccgccgggagcggattt gaacgttgcg aagcaacggc 960 ccggagggtg gcgggcagga cgcccgccataaactgccag gcatcaaatt aagcagaagg 1020 ccatcctgac ggatggcctt tttgcgtttctacaaactct tttgtttatt tttctaaata 1080 cattcaaata tgtatccgct catgagacaataaccctgat aaatgcttca ataatattga 1140 aaaaggaaga gtatgagtat tcaacatttccgtgtcgccc ttattccctt ttttgcggca 1200 ttttgccttc ctgtttttgc tcacccagaaacgctggtga aagtaaaaga tgctgaagat 1260 cagttgggtg cacgagtggg ttacatcgaactggatctca acagcggtaa gatccttgag 1320 agttttcgcc ccgaagaacg ttttccaatgatgagcactt ttaaagttct gctatgtggc 1380 gcggtattat cccgtgttga cgccgggcaagagcaactcg gtcgccgcat acactattct 1440 cagaatgact tggttgagta ctcaccagtcacagaaaagc atcttacgga tggcatgaca 1500 gtaagagaat tatgcagtgc tgccataaccatgagtgata acactgcggc caacttactt 1560 ctgacaacga tcggaggacc gaaggagctaaccgcttttt tgcacaacat gggggatcat 1620 gtaactcgcc atgatcgttg ggaaccggagctgaatgaag ccataccaaa cgacgagcgt 1680 gacaccacga tgcctgtagc aatggcaacaacgttgcgca aactattaac tggcgaacta 1740 cttactctag cttcccggca acaattaatagactggatgg aggcggataa agttgcagga 1800 ccacttctgc gctcggccct tccggctggctggtttattg ctgataaatc tggagccggt 1860 gagcgtgggt ctcgcggtat cattgcagcactggggccag atggtaagcc ctcccgtatc 1920 gtagttatct acacgacggg gagtcaggcaactatggatg aacgaaatag acagatcgct 1980 gagataggtg cctcactgat taagcattggtaactgtcag accaagttta ctcatatata 2040 ctttagattg atttaaaact tcatttttaatttaaaagga tctaggtgaa gatccttttt 2100 gataatctca tgaccaaaat cccttaacgtgagttttcgt tccactgagc gtcagacccc 2160 gtagaaaaga tcaaaggatc ttcttgagatcctttttttc tgcgcgtaat ctgctgcttg 2220 caaacaaaaa aaccaccgct accagcggtggtttgtttgc cggatcaaga gctaccaact 2280 ctttttccga aggtaactgg cttcagcagagcgcagatac caaatactgt ccttctagtg 2340 tagccgtagt taggccacca cttcaagaactctgtagcac cgcctacata cctcgctctg 2400 ctaatcctgt taccagtggc tgctgccagtggcgataagt cgtgtcttac cgggttggac 2460 tcaagacgat agttaccgga taaggcgcagcggtcgggct gaacgggggg ttcgtgcaca 2520 cagcccagct tggagcgaac gacctacaccgaactgagat acctacagcg tgagcattga 2580 gaaagcgcca cgcttcccga agggagaaaggcggacaggt atccggtaag cggcagggtc 2640 ggaacaggag agcgcacgag ggagcttccagggggaaacg cctggtatct ttatagtcct 2700 gtcgggtttc gccacctctg acttgagcgtcgatttttgt gatgctcgtc aggggggcgg 2760 agcctatgga aaaacgccag caacgcggcctttttacggt tcctggcctt ttgctggcct 2820 tttgctcaca tgttctttcc tgcgttatcccctgattctg tggataaccg tattaccgcc 2880 tttgagtgag ctgataccgc tcgccgcagccgaacgaccg agcgcagcga gtcagtgagc 2940 gaggaagcgg aagagcgcct gatgcggtattttctcctta cgcatctgtg cggtatttca 3000 caccgcagat cctgacgcgc cctgtagcggcgcattaagc gcggcgggtg tggtggttac 3060 gcgcagcgtg accgctacac ttgccagcgccctagcgccc gctcctttcg ctttcttccc 3120 ttcctttctc gccacgttcg ccggctttccccgtcaagct ctaaatcggg ggctcccttt 3180 agggttccga tttagtgctt tacggcacctcgaccccaaa aaacttgatt agggtgatgg 3240 ttcacgtagt gggccatcgc cctgatagacggtttttcgc cctttgacgt tggagtccac 3300 gttctttaat agtggactct tgttccaaactggaacaaca ctcaacccta tctcggtcta 3360 ttcttttgat ttataaggga ttttgccgatttcggcctat tggttaaaaa atgagctgat 3420 ttaacaaaaa tttaacgcga attttaacaaaatattaacg tttacaggat ctaatgaggg 3480 gacgacgaca gtatcggcct caggaagatcgcactccagc cagctttccg gcaccgcttc 3540 tggtgccgga aaccaggcaa agcgccattcgccattcagg ctgcgcaact gttgggaagg 3600 gcgatcggtg cgggcctctt cgctattacgccagctggcg aaagggggat gtgctgcaag 3660 gcgattaagt tgggtaacgc cagggttttcccagtcacga cgttgtaaaa cgacggccag 3720 tgaatccgta atcatggtca tagctgtttcctgtgtgaaa ttgttatccg ctcacaattc 3780 cacacaacat acgagccgga agcataaagtgtaaagcctg gggtgcctaa tgagtgagct 3840 aactcacatt aattgcgttg cgctcactgcccgctttcca gtcgggaaac ctgtcgtgcc 3900 agctgcatta atgaatcggc caacgcgcggggagaggcgg tttgcgtatt gggcgccagg 3960 gtggtttttc ttttcaccag tgagacgggcaacagctgat tgcccttcac cgcctggccc 4020 tgagagagtt gcagcaagcg gtccacgctggtttgcccca gcaggcgaaa atcctgtttg 4080 atggtggttg acggcgggat ataacatgagctgtcttcgg tatcgtcgta tcccactacc 4140 gagatatccg caccaacgcg cagcccggactcggtaatgg cgcgcattgc gcccagcgcc 4200 atctgatcgt tggcaaccag catcgcagtgggaacgatgc cctcattcag catttgcatg 4260 gtttgttgaa aaccggacat ggcactccagtcgccttccc gttccgctat cggctgaatt 4320 tgattgcgag tgagatattt atgccagccagccagacgca gacgcgccga gacagaactt 4380 aatgggcccg ctaacagcgc gatttgctggtgacccaatg cgaccagatg ctccacgccc 4440 agtcgcgtac cgtcttcatg ggagaaaataatactgttga tgggtgtctg gtcagagaca 4500 tcaagaaata acgccggaac attagtgcaggcagcttcca cagcaatggc atcctggtca 4560 tccagcggat agttaatgat cagcccactgacgcgttgcg cgagaagatt gtgcaccgcc 4620 gctttacagg cttcgacgcc gcttcgttctaccatcgaca ccaccacgct ggcacccagt 4680 tgatcggcgc gagatttaat cgccgcgacaatttgcgacg gcgcgtgcag ggccagactg 4740 gaggtggcaa cgccaatcag caacgactgtttgcccgcca gttgttgtgc cacgcggttg 4800 ggaatgtaat tcagctccgc catcgccgcttccacttttt cccgcgtttt cgcagaaacg 4860 tggctggcct ggttcaccac gcgggaaacggtctgataag agacaccggc atactctgcg 4920 acatcgtata acgttactgg tttcacattcaccaccctga attgactctc ttccgggcgc 4980 tatcatgcca taccgcgaaa ggttttgcaccattccatgg tgtcgaattg ctgcaggtcg 5040 agggggtcat ggctgcgccc cgacacccgccaacacccgc tgacgcgccc tgacgggctt 5100 gtctgctccc ggcatccgct tacagacaagctgtgaccgt ctccgggagc tgcatgtgtc 5160 agaggttttc accgtcatca ccgaaacgcgcgaggcagca aggagatggc gcccaacagt 5220 cccccggcca cgggcctgcc accatacccacgccgaaaca agcgctcatg agcccgaagt 5280 ggcgagcccg atcttcccca tcggtgatgtcggcgatata ggcgccagca accgcacctg 5340 tggcgccggt gatgccggcc acgatgcgtccggcgtagag gatccggagc ttatcgactg 5400 cacggtgcac caatgcttct ggcgtcaggcagccatcgga agctgtggta tggctgtgca 5460 ggtcgtaaat cactgcataa ttcgtgtcgctcaaggcgca ctcccgttct ggataatgtt 5520 ttttgcgccg a 5531 20 3897 DNAArtificial sequence pNCO-His6-BS-LuSy Expression vector 20 ctcgagaaatcataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca 60 attgtgagcggataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaat 120 atcatacaaggaaatttagt tggtacaggt cttaaaatcg gaatcgtagt aggaagattt 180 aatgattttattacgagcaa gctgctgagc ggagcagaag atgcgctgct cagacatggc 240 gtagacacaaatgacattga tgtggcttgg gttccaggcg catttgaaat accgtttgct 300 gcgaaaaaaatggcggaaac aaaaaaatat gatgctatta tcacattggg cactgtcatc 360 agaggcgcaacgacacatta cgattatgtc tgcaatgaag ctgcaaaagg catcgcgcaa 420 gcagcaaacactactggtgt acctgtcatc tttggaattg taacaactga aaacatcgaa 480 caggctatcgagcgtgccgg cacaaaagcg ggcaacaaag gtgtagattg tgctgtttct 540 gccattgaaatggcaaactt aaaccgttct ttcgaacatc accatcacca ccattaagga 600 tccgtcgacctgcagccaag cttaattagc tgagcttgga ctcctgttga tagatccagt 660 aatgacctcagaactccatc tggatttgtt cagaacgctc ggttgccgcc gggcgttttt 720 tattggtgagaatccaagct agcttggcga gattttcagg agctaaggaa gctaaaatgg 780 agaaaaaaatcactggatat accaccgttg atatatccca atggcatcgt aaagaacatt 840 ttgaggcatttcagtcagtt gctcaatgta cctataacca gaccgttcag ctggatatta 900 cggcctttttaaagaccgta aagaaaaata agcacaagtt ttatccggcc tttattcaca 960 ttcttgcccgcctgatgaat gctcatccgg aatttcgtat ggcaatgaaa gacggtgagc 1020 tggtgatatgggatagtgtt cacccttgtt acaccgtttt ccatgagcaa actgaaacgt 1080 tttcatcgctctggagtgaa taccacgacg atttccggca gtttctacac atatattcgc 1140 aagatgtggcgtgttacggt gaaaacctgg cctatttccc taaagggttt attgagaata 1200 tgtttttcgtctcagccaat ccctgggtga gtttcaccag ttttgattta aacgtggcca 1260 atatggacaacttcttcgcc cccgttttca ccatgcatgg gcaaatatta tacgcaaggc 1320 gacaaggtgctgatgccgct ggcgattcag gttcatcatg ccgtctgtga tggcttccat 1380 gtcggcagaatgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa 1440 tttttttaaggcagttattg gtgcccttaa acgcctgggg taatgactct ctagcttgag 1500 gcatcaaataaaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt 1560 gtcggtgaacgctctcctga gtaggacaaa tccgccgctc tagagctgcc tcgcgcgttt 1620 cggtgatgacggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct 1680 gtaagcggatgccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg 1740 tcggggcgcagccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat 1800 gcggcatcagagcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga 1860 tgcgtaaggagaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg 1920 cgctcggtctgtcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 1980 tccacagaatcaggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 2040 aggaaccgtaaaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 2100 catcacaaaaatcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 2160 caggcgtttccccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 2220 ggatacctgtccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt 2280 aggtatctcagttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 2340 gttcagcccgaccgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 2400 cacgacttatcgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 2460 ggcggtgctacagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 2520 tttggtatctgcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 2580 tccggcaaacaaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 2640 cgcagaaaaaaaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 2700 tggaacgaaaactcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 2760 tagatccttttaaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 2820 tggtctgacagttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 2880 cgttcatccatagctgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 2940 ccatctggccccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 3000 tcagcaataaaccagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 3060 gcctccatccagtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 3120 agtttgcgcaacgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 3180 atggcttcattcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 3240 tgcaaaaaagcggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 3300 gtgttatcactcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 3360 agatgcttttctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 3420 cgaccgagttgctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 3480 ttaaaagtgctcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 3540 ctgttgagatccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 3600 actttcaccagcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 3660 ataagggcgacacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 3720 atttatcagggttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 3780 caaataggggttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt 3840 attatcatgacattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcac 3897 21 3879 DNAArtificial sequence pNCO-AA-LuSy Expression vector 21 ctcgagaaatcataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca 60 attgtgagcggataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgcaa 120 atctacgaaggtaaactaac tgctgaaggc cttcgtttcg gtatcgtagc atcacgtttt 180 aatcatgctcttgtcgaccg tctggtggag ggtgcaattg attgcatagt ccgtcatggc 240 ggccgtgaagaagacattac tctggttcgt gttccaggct catgggaaat accggttgct 300 gcgggtgaactggcgcgtaa agaggacatt gatgctgtta tcgcaattgg cgttctcatc 360 agaggcgcaacgccacattt cgattatatc gcctctgaag tttcaaaagg cctcgcgaac 420 ctttcattagaactacgtaa acctatcacc ttcggtgtta ttacagctga caccttggaa 480 caggctatcgagcgcgccgg cacaaaacac ggcaacaaag gttgggaagc agcgctttct 540 gccattgaaatggcaaactt attcaagtct ctccgataag gatccgtcga cctgcagcca 600 agcttaattagctgagcttg gactcctgtt gatagatcca gtaatgacct cagaactcca 660 tctggatttgttcagaacgc tcggttgccg ccgggcgttt tttattggtg agaatccaag 720 ctagcttggcgagattttca ggagctaagg aagctaaaat ggagaaaaaa atcactggat 780 ataccaccgttgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcag 840 ttgctcaatgtacctataac cagaccgttc agctggatat tacggccttt ttaaagaccg 900 taaagaaaaataagcacaag ttttatccgg cctttattca cattcttgcc cgcctgatga 960 atgctcatccggaatttcgt atggcaatga aagacggtga gctggtgata tgggatagtg 1020 ttcacccttgttacaccgtt ttccatgagc aaactgaaac gttttcatcg ctctggagtg 1080 aataccacgacgatttccgg cagtttctac acatatattc gcaagatgtg gcgtgttacg 1140 gtgaaaacctggcctatttc cctaaagggt ttattgagaa tatgtttttc gtctcagcca 1200 atccctgggtgagtttcacc agttttgatt taaacgtggc caatatggac aacttcttcg 1260 cccccgttttcaccatgcat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg 1320 ctggcgattcaggttcatca tgccgtctgt gatggcttcc atgtcggcag aatgcttaat 1380 gaattacaacagtactgcga tgagtggcag ggcggggcgt aattttttta aggcagttat 1440 tggtgcccttaaacgcctgg ggtaatgact ctctagcttg aggcatcaaa taaaacgaaa 1500 ggctcagtcgaaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct 1560 gagtaggacaaatccgccgc tctagagctg cctcgcgcgt ttcggtgatg acggtgaaaa 1620 cctctgacacatgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag 1680 cagacaagcccgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac 1740 ccagtcacgtagcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt 1800 gtactgagagtgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac 1860 cgcatcaggcgctcttccgc ttcctcgctc actgactcgc tgcgctcggt ctgtcggctg 1920 cggcgagcggtatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 1980 aacgcaggaaagaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 2040 gcgttgctggcgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 2100 tcaagtcagaggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 2160 agctccctcgtgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 2220 ctcccttcgggaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg 2280 taggtcgttcgctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 2340 gccttatccggtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 2400 gcagcagccactggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 2460 ttgaagtggtggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg 2520 ctgaagccagttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 2580 gctggtagcggtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 2640 caagaagatcctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 2700 taagggattttggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 2760 aaatgaagttttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 2820 tgcttaatcagtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagctgcc 2880 tgactccccgtcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 2940 gcaatgataccgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 3000 gccggaagggccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 3060 aattgttgccgggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 3120 gccattgctacaggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 3180 ggttcccaacgatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 3240 tccttcggtcctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 3300 atggcagcactgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 3360 ggtgagtactcaaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 3420 ccggcgtcaatacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 3480 ggaaaacgttcttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 3540 atgtaacccactcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 3600 gggtgagcaaaaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 3660 tgttgaatactcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 3720 ctcatgagcggatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 3780 acatttccccgaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc 3840 tataaaaataggcgtatcac gaggcccttt cgtcttcac 3879 22 3927 DNA Artificial sequencepNCO-C-Biotag-AA-LuSy Expression vector 22 ctcgagaaat cataaaaaatttatttgctt tgtgagcgga taacaattat aatagattca 60 attgtgagcg gataacaatttcacacagaa ttcattaaag aggagaaatt aactatgcaa 120 atctacgaag gtaaactaactgctgaaggc cttcgtttcg gtatcgtagc atcacgtttt 180 aatcatgctc ttgtcgaccgtctggtggag ggtgcaattg attgcatagt ccgtcatggc 240 ggccgtgaag aagacattactctggttcgt gttccaggct catgggaaat accggttgct 300 gcgggtgaac tggcgcgtaaagaggacatt gatgctgtta tcgcaattgg cgttctcatc 360 agaggcgcaa cgccacatttcgattatatc gcctctgaag tttcaaaagg cctcgcgaac 420 ctttcattag aactacgtaaacctatcacc ttcggtgtta ttacagctga caccttggaa 480 caggctatcg agcgcgccggcacaaaacac ggcaacaaag gttgggaagc agcgctttct 540 gccattgaaa tggcaaacttattcaagtct ctccgagcgg ccgcactcgg cggcatcttc 600 gaagctatga agatggagtggcgctaagga tccgtcgacc tgcagccaag cttaattagc 660 tgagcttgga ctcctgttgatagatccagt aatgacctca gaactccatc tggatttgtt 720 cagaacgctc ggttgccgccgggcgttttt tattggtgag aatccaagct agcttggcga 780 gattttcagg agctaaggaagctaaaatgg agaaaaaaat cactggatat accaccgttg 840 atatatccca atggcatcgtaaagaacatt ttgaggcatt tcagtcagtt gctcaatgta 900 cctataacca gaccgttcagctggatatta cggccttttt aaagaccgta aagaaaaata 960 agcacaagtt ttatccggcctttattcaca ttcttgcccg cctgatgaat gctcatccgg 1020 aatttcgtat ggcaatgaaagacggtgagc tggtgatatg ggatagtgtt cacccttgtt 1080 acaccgtttt ccatgagcaaactgaaacgt tttcatcgct ctggagtgaa taccacgacg 1140 atttccggca gtttctacacatatattcgc aagatgtggc gtgttacggt gaaaacctgg 1200 cctatttccc taaagggtttattgagaata tgtttttcgt ctcagccaat ccctgggtga 1260 gtttcaccag ttttgatttaaacgtggcca atatggacaa cttcttcgcc cccgttttca 1320 ccatgcatgg gcaaatattatacgcaaggc gacaaggtgc tgatgccgct ggcgattcag 1380 gttcatcatg ccgtctgtgatggcttccat gtcggcagaa tgcttaatga attacaacag 1440 tactgcgatg agtggcagggcggggcgtaa tttttttaag gcagttattg gtgcccttaa 1500 acgcctgggg taatgactctctagcttgag gcatcaaata aaacgaaagg ctcagtcgaa 1560 agactgggcc tttcgttttatctgttgttt gtcggtgaac gctctcctga gtaggacaaa 1620 tccgccgctc tagagctgcctcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat 1680 gcagctcccg gagacggtcacagcttgtct gtaagcggat gccgggagca gacaagcccg 1740 tcagggcgcg tcagcgggtgttggcgggtg tcggggcgca gccatgaccc agtcacgtag 1800 cgatagcgga gtgtatactggcttaactat gcggcatcag agcagattgt actgagagtg 1860 caccatatgc ggtgtgaaataccgcacaga tgcgtaagga gaaaataccg catcaggcgc 1920 tcttccgctt cctcgctcactgactcgctg cgctcggtct gtcggctgcg gcgagcggta 1980 tcagctcact caaaggcggtaatacggtta tccacagaat caggggataa cgcaggaaag 2040 aacatgtgag caaaaggccagcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 2100 tttttccata ggctccgcccccctgacgag catcacaaaa atcgacgctc aagtcagagg 2160 tggcgaaacc cgacaggactataaagatac caggcgtttc cccctggaag ctccctcgtg 2220 cgctctcctg ttccgaccctgccgcttacc ggatacctgt ccgcctttct cccttcggga 2280 agcgtggcgc tttctcaatgctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 2340 tccaagctgg gctgtgtgcacgaacccccc gttcagcccg accgctgcgc cttatccggt 2400 aactatcgtc ttgagtccaacccggtaaga cacgacttat cgccactggc agcagccact 2460 ggtaacagga ttagcagagcgaggtatgta ggcggtgcta cagagttctt gaagtggtgg 2520 cctaactacg gctacactagaaggacagta tttggtatct gcgctctgct gaagccagtt 2580 accttcggaa aaagagttggtagctcttga tccggcaaac aaaccaccgc tggtagcggt 2640 ggtttttttg tttgcaagcagcagattacg cgcagaaaaa aaggatctca agaagatcct 2700 ttgatctttt ctacggggtctgacgctcag tggaacgaaa actcacgtta agggattttg 2760 gtcatgagat tatcaaaaaggatcttcacc tagatccttt taaattaaaa atgaagtttt 2820 aaatcaatct aaagtatatatgagtaaact tggtctgaca gttaccaatg cttaatcagt 2880 gaggcaccta tctcagcgatctgtctattt cgttcatcca tagctgcctg actccccgtc 2940 gtgtagataa ctacgatacgggagggctta ccatctggcc ccagtgctgc aatgataccg 3000 cgagacccac gctcaccggctccagattta tcagcaataa accagccagc cggaagggcc 3060 gagcgcagaa gtggtcctgcaactttatcc gcctccatcc agtctattaa ttgttgccgg 3120 gaagctagag taagtagttcgccagttaat agtttgcgca acgttgttgc cattgctaca 3180 ggcatcgtgg tgtcacgctcgtcgtttggt atggcttcat tcagctccgg ttcccaacga 3240 tcaaggcgag ttacatgatcccccatgttg tgcaaaaaag cggttagctc cttcggtcct 3300 ccgatcgttg tcagaagtaagttggccgca gtgttatcac tcatggttat ggcagcactg 3360 cataattctc ttactgtcatgccatccgta agatgctttt ctgtgactgg tgagtactca 3420 accaagtcat tctgagaatagtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 3480 cgggataata ccgcgccacatagcagaact ttaaaagtgc tcatcattgg aaaacgttct 3540 tcggggcgaa aactctcaaggatcttaccg ctgttgagat ccagttcgat gtaacccact 3600 cgtgcaccca actgatcttcagcatctttt actttcacca gcgtttctgg gtgagcaaaa 3660 acaggaaggc aaaatgccgcaaaaaaggga ataagggcga cacggaaatg ttgaatactc 3720 atactcttcc tttttcaatattattgaagc atttatcagg gttattgtct catgagcgga 3780 tacatatttg aatgtatttagaaaaataaa caaatagggg ttccgcgcac atttccccga 3840 aaagtgccac ctgacgtctaagaaaccatt attatcatga cattaaccta taaaaatagg 3900 cgtatcacga ggccctttcgtcttcac 3927 23 3945 DNA Artificial sequence pNCO-His6-C-Biotag-AA-LuSyExpression vector 23 ctcgagaaat cataaaaaat ttatttgctt tgtgagcggataacaattat aatagattca 60 attgtgagcg gataacaatt tcacacagaa ttcattaaagaggagaaatt aactatgcaa 120 atctacgaag gtaaactaac tgctgaaggc cttcgtttcggtatcgtagc atcacgtttt 180 aatcatgctc ttgtcgaccg tctggtggag ggtgcaattgattgcatagt ccgtcatggc 240 ggccgtgaag aagacattac tctggttcgt gttccaggctcatgggaaat accggttgct 300 gcgggtgaac tggcgcgtaa agaggacatt gatgctgttatcgcaattgg cgttctcatc 360 agaggcgcaa cgccacattt cgattatatc gcctctgaagtttcaaaagg cctcgcgaac 420 ctttcattag aactacgtaa acctatcacc ttcggtgttattacagctga caccttggaa 480 caggctatcg agcgcgccgg cacaaaacac ggcaacaaaggttgggaagc agcgctttct 540 gccattgaaa tggcaaactt attcaagtct ctccgacatcaccatcacca ccatgcggcc 600 gcactcggcg gcatcttcga agctatgaag atggagtggcgctaaggatc cgtcgacctg 660 cagccaagct taattagctg agcttggact cctgttgatagatccagtaa tgacctcaga 720 actccatctg gatttgttca gaacgctcgg ttgccgccgggcgtttttta ttggtgagaa 780 tccaagctag cttggcgaga ttttcaggag ctaaggaagctaaaatggag aaaaaaatca 840 ctggatatac caccgttgat atatcccaat ggcatcgtaaagaacatttt gaggcatttc 900 agtcagttgc tcaatgtacc tataaccaga ccgttcagctggatattacg gcctttttaa 960 agaccgtaaa gaaaaataag cacaagtttt atccggcctttattcacatt cttgcccgcc 1020 tgatgaatgc tcatccggaa tttcgtatgg caatgaaagacggtgagctg gtgatatggg 1080 atagtgttca cccttgttac accgttttcc atgagcaaactgaaacgttt tcatcgctct 1140 ggagtgaata ccacgacgat ttccggcagt ttctacacatatattcgcaa gatgtggcgt 1200 gttacggtga aaacctggcc tatttcccta aagggtttattgagaatatg tttttcgtct 1260 cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaacgtggccaat atggacaact 1320 tcttcgcccc cgttttcacc atgcatgggc aaatattatacgcaaggcga caaggtgctg 1380 atgccgctgg cgattcaggt tcatcatgcc gtctgtgatggcttccatgt cggcagaatg 1440 cttaatgaat tacaacagta ctgcgatgag tggcagggcggggcgtaatt tttttaaggc 1500 agttattggt gcccttaaac gcctggggta atgactctctagcttgaggc atcaaataaa 1560 acgaaaggct cagtcgaaag actgggcctt tcgttttatctgttgtttgt cggtgaacgc 1620 tctcctgagt aggacaaatc cgccgctcta gagctgcctcgcgcgtttcg gtgatgacgg 1680 tgaaaacctc tgacacatgc agctcccgga gacggtcacagcttgtctgt aagcggatgc 1740 cgggagcaga caagcccgtc agggcgcgtc agcgggtgttggcgggtgtc ggggcgcagc 1800 catgacccag tcacgtagcg atagcggagt gtatactggcttaactatgc ggcatcagag 1860 cagattgtac tgagagtgca ccatatgcgg tgtgaaataccgcacagatg cgtaaggaga 1920 aaataccgca tcaggcgctc ttccgcttcc tcgctcactgactcgctgcg ctcggtctgt 1980 cggctgcggc gagcggtatc agctcactca aaggcggtaatacggttatc cacagaatca 2040 ggggataacg caggaaagaa catgtgagca aaaggccagcaaaaggccag gaaccgtaaa 2100 aaggccgcgt tgctggcgtt tttccatagg ctccgcccccctgacgagca tcacaaaaat 2160 cgacgctcaa gtcagaggtg gcgaaacccg acaggactataaagatacca ggcgtttccc 2220 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgccgcttaccgg atacctgtcc 2280 gcctttctcc cttcgggaag cgtggcgctt tctcaatgctcacgctgtag gtatctcagt 2340 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacgaaccccccgt tcagcccgac 2400 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacccggtaagaca cgacttatcg 2460 ccactggcag cagccactgg taacaggatt agcagagcgaggtatgtagg cggtgctaca 2520 gagttcttga agtggtggcc taactacggc tacactagaaggacagtatt tggtatctgc 2580 gctctgctga agccagttac cttcggaaaa agagttggtagctcttgatc cggcaaacaa 2640 accaccgctg gtagcggtgg tttttttgtt tgcaagcagcagattacgcg cagaaaaaaa 2700 ggatctcaag aagatccttt gatcttttct acggggtctgacgctcagtg gaacgaaaac 2760 tcacgttaag ggattttggt catgagatta tcaaaaaggatcttcaccta gatcctttta 2820 aattaaaaat gaagttttaa atcaatctaa agtatatatgagtaaacttg gtctgacagt 2880 taccaatgct taatcagtga ggcacctatc tcagcgatctgtctatttcg ttcatccata 2940 gctgcctgac tccccgtcgt gtagataact acgatacgggagggcttacc atctggcccc 3000 agtgctgcaa tgataccgcg agacccacgc tcaccggctccagatttatc agcaataaac 3060 cagccagccg gaagggccga gcgcagaagt ggtcctgcaactttatccgc ctccatccag 3120 tctattaatt gttgccggga agctagagta agtagttcgccagttaatag tttgcgcaac 3180 gttgttgcca ttgctacagg catcgtggtg tcacgctcgtcgtttggtat ggcttcattc 3240 agctccggtt cccaacgatc aaggcgagtt acatgatcccccatgttgtg caaaaaagcg 3300 gttagctcct tcggtcctcc gatcgttgtc agaagtaagttggccgcagt gttatcactc 3360 atggttatgg cagcactgca taattctctt actgtcatgccatccgtaag atgcttttct 3420 gtgactggtg agtactcaac caagtcattc tgagaatagtgtatgcggcg accgagttgc 3480 tcttgcccgg cgtcaatacg ggataatacc gcgccacatagcagaacttt aaaagtgctc 3540 atcattggaa aacgttcttc ggggcgaaaa ctctcaaggatcttaccgct gttgagatcc 3600 agttcgatgt aacccactcg tgcacccaac tgatcttcagcatcttttac tttcaccagc 3660 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaaaaaagggaat aagggcgaca 3720 cggaaatgtt gaatactcat actcttcctt tttcaatattattgaagcat ttatcagggt 3780 tattgtctca tgagcggata catatttgaa tgtatttagaaaaataaaca aataggggtt 3840 ccgcgcacat ttccccgaaa agtgccacct gacgtctaagaaaccattat tatcatgaca 3900 ttaacctata aaaataggcg tatcacgagg ccctttcgtcttcac 3945 24 3957 DNA Artificial sequence pNCO-His6-Gly2-Ser-Gly-C-Biotag-AA-LuSy Expression vector 24 ctcgagaaat cataaaaaat ttatttgctttgtgagcgga taacaattat aatagattca 60 attgtgagcg gataacaatt tcacacagaattcattaaag aggagaaatt aactatgcaa 120 atctacgaag gtaaactaac tgctgaaggccttcgtttcg gtatcgtagc atcacgtttt 180 aatcatgctc ttgtcgaccg tctggtggagggtgcaattg attgcatagt ccgtcatggc 240 ggccgtgaag aagacattac tctggttcgtgttccaggct catgggaaat accggttgct 300 gcgggtgaac tggcgcgtaa agaggacattgatgctgtta tcgcaattgg cgttctcatc 360 agaggcgcaa cgccacattt cgattatatcgcctctgaag tttcaaaagg cctcgcgaac 420 ctttcattag aactacgtaa acctatcaccttcggtgtta ttacagctga caccttggaa 480 caggctatcg agcgcgccgg cacaaaacacggcaacaaag gttgggaagc agcgctttct 540 gccattgaaa tggcaaactt attcaagtctctccgacatc accatcacca ccatggcggt 600 tctggcgcgg ccgcactcgg cggcatcttcgaagctatga agatggagtg gcgctaagga 660 tccgtcgacc tgcagccaag cttaattagctgagcttgga ctcctgttga tagatccagt 720 aatgacctca gaactccatc tggatttgttcagaacgctc ggttgccgcc gggcgttttt 780 tattggtgag aatccaagct agcttggcgagattttcagg agctaaggaa gctaaaatgg 840 agaaaaaaat cactggatat accaccgttgatatatccca atggcatcgt aaagaacatt 900 ttgaggcatt tcagtcagtt gctcaatgtacctataacca gaccgttcag ctggatatta 960 cggccttttt aaagaccgta aagaaaaataagcacaagtt ttatccggcc tttattcaca 1020 ttcttgcccg cctgatgaat gctcatccggaatttcgtat ggcaatgaaa gacggtgagc 1080 tggtgatatg ggatagtgtt cacccttgttacaccgtttt ccatgagcaa actgaaacgt 1140 tttcatcgct ctggagtgaa taccacgacgatttccggca gtttctacac atatattcgc 1200 aagatgtggc gtgttacggt gaaaacctggcctatttccc taaagggttt attgagaata 1260 tgtttttcgt ctcagccaat ccctgggtgagtttcaccag ttttgattta aacgtggcca 1320 atatggacaa cttcttcgcc cccgttttcaccatgcatgg gcaaatatta tacgcaaggc 1380 gacaaggtgc tgatgccgct ggcgattcaggttcatcatg ccgtctgtga tggcttccat 1440 gtcggcagaa tgcttaatga attacaacagtactgcgatg agtggcaggg cggggcgtaa 1500 tttttttaag gcagttattg gtgcccttaaacgcctgggg taatgactct ctagcttgag 1560 gcatcaaata aaacgaaagg ctcagtcgaaagactgggcc tttcgtttta tctgttgttt 1620 gtcggtgaac gctctcctga gtaggacaaatccgccgctc tagagctgcc tcgcgcgttt 1680 cggtgatgac ggtgaaaacc tctgacacatgcagctcccg gagacggtca cagcttgtct 1740 gtaagcggat gccgggagca gacaagcccgtcagggcgcg tcagcgggtg ttggcgggtg 1800 tcggggcgca gccatgaccc agtcacgtagcgatagcgga gtgtatactg gcttaactat 1860 gcggcatcag agcagattgt actgagagtgcaccatatgc ggtgtgaaat accgcacaga 1920 tgcgtaagga gaaaataccg catcaggcgctcttccgctt cctcgctcac tgactcgctg 1980 cgctcggtct gtcggctgcg gcgagcggtatcagctcact caaaggcggt aatacggtta 2040 tccacagaat caggggataa cgcaggaaagaacatgtgag caaaaggcca gcaaaaggcc 2100 aggaaccgta aaaaggccgc gttgctggcgtttttccata ggctccgccc ccctgacgag 2160 catcacaaaa atcgacgctc aagtcagaggtggcgaaacc cgacaggact ataaagatac 2220 caggcgtttc cccctggaag ctccctcgtgcgctctcctg ttccgaccct gccgcttacc 2280 ggatacctgt ccgcctttct cccttcgggaagcgtggcgc tttctcaatg ctcacgctgt 2340 aggtatctca gttcggtgta ggtcgttcgctccaagctgg gctgtgtgca cgaacccccc 2400 gttcagcccg accgctgcgc cttatccggtaactatcgtc ttgagtccaa cccggtaaga 2460 cacgacttat cgccactggc agcagccactggtaacagga ttagcagagc gaggtatgta 2520 ggcggtgcta cagagttctt gaagtggtggcctaactacg gctacactag aaggacagta 2580 tttggtatct gcgctctgct gaagccagttaccttcggaa aaagagttgg tagctcttga 2640 tccggcaaac aaaccaccgc tggtagcggtggtttttttg tttgcaagca gcagattacg 2700 cgcagaaaaa aaggatctca agaagatcctttgatctttt ctacggggtc tgacgctcag 2760 tggaacgaaa actcacgtta agggattttggtcatgagat tatcaaaaag gatcttcacc 2820 tagatccttt taaattaaaa atgaagttttaaatcaatct aaagtatata tgagtaaact 2880 tggtctgaca gttaccaatg cttaatcagtgaggcaccta tctcagcgat ctgtctattt 2940 cgttcatcca tagctgcctg actccccgtcgtgtagataa ctacgatacg ggagggctta 3000 ccatctggcc ccagtgctgc aatgataccgcgagacccac gctcaccggc tccagattta 3060 tcagcaataa accagccagc cggaagggccgagcgcagaa gtggtcctgc aactttatcc 3120 gcctccatcc agtctattaa ttgttgccgggaagctagag taagtagttc gccagttaat 3180 agtttgcgca acgttgttgc cattgctacaggcatcgtgg tgtcacgctc gtcgtttggt 3240 atggcttcat tcagctccgg ttcccaacgatcaaggcgag ttacatgatc ccccatgttg 3300 tgcaaaaaag cggttagctc cttcggtcctccgatcgttg tcagaagtaa gttggccgca 3360 gtgttatcac tcatggttat ggcagcactgcataattctc ttactgtcat gccatccgta 3420 agatgctttt ctgtgactgg tgagtactcaaccaagtcat tctgagaata gtgtatgcgg 3480 cgaccgagtt gctcttgccc ggcgtcaatacgggataata ccgcgccaca tagcagaact 3540 ttaaaagtgc tcatcattgg aaaacgttcttcggggcgaa aactctcaag gatcttaccg 3600 ctgttgagat ccagttcgat gtaacccactcgtgcaccca actgatcttc agcatctttt 3660 actttcacca gcgtttctgg gtgagcaaaaacaggaaggc aaaatgccgc aaaaaaggga 3720 ataagggcga cacggaaatg ttgaatactcatactcttcc tttttcaata ttattgaagc 3780 atttatcagg gttattgtct catgagcggatacatatttg aatgtattta gaaaaataaa 3840 caaatagggg ttccgcgcac atttccccgaaaagtgccac ctgacgtcta agaaaccatt 3900 attatcatga cattaaccta taaaaataggcgtatcacga ggccctttcg tcttcac 3957 25 3879 DNA Artificial sequencepNCO-BS-LuSy-AgeI-AA-LuSy Expression vector 25 ctcgagaaat cataaaaaatttatttgctt tgtgagcgga taacaattat aatagattca 60 attgtgagcg gataacaatttcacacagaa ttcattaaag aggagaaatt aactatgaat 120 atcatacaag gaaatttagttggtacaggt cttaaaatcg gaatcgtagt aggaagattt 180 aatgatttta ttacgagcaagctgctgagc ggagcagaag atgcgctgct cagacatggc 240 gtagacacaa atgacattgatgtggcttgg gttccaggcg catttgaaat accggttgct 300 gcgggtgaac tggcgcgtaaagaggacatt gatgctgtta tcgcaattgg cgttctcatc 360 agaggcgcaa cgccacatttcgattatatc gcctctgaag tttcaaaagg cctcgcgaac 420 ctttcattag aactacgtaaacctatcacc ttcggtgtta ttacagctga caccttggaa 480 caggctatcg agcgcgccggcacaaaacac ggcaacaaag gttgggaagc agcgctttct 540 gccattgaaa tggcaaacttattcaagtct ctccgataag gatccgtcga cctgcagcca 600 agcttaatta gctgagcttggactcctgtt gatagatcca gtaatgacct cagaactcca 660 tctggatttg ttcagaacgctcggttgccg ccgggcgttt tttattggtg agaatccaag 720 ctagcttggc gagattttcaggagctaagg aagctaaaat ggagaaaaaa atcactggat 780 ataccaccgt tgatatatcccaatggcatc gtaaagaaca ttttgaggca tttcagtcag 840 ttgctcaatg tacctataaccagaccgttc agctggatat tacggccttt ttaaagaccg 900 taaagaaaaa taagcacaagttttatccgg cctttattca cattcttgcc cgcctgatga 960 atgctcatcc ggaatttcgtatggcaatga aagacggtga gctggtgata tgggatagtg 1020 ttcacccttg ttacaccgttttccatgagc aaactgaaac gttttcatcg ctctggagtg 1080 aataccacga cgatttccggcagtttctac acatatattc gcaagatgtg gcgtgttacg 1140 gtgaaaacct ggcctatttccctaaagggt ttattgagaa tatgtttttc gtctcagcca 1200 atccctgggt gagtttcaccagttttgatt taaacgtggc caatatggac aacttcttcg 1260 cccccgtttt caccatgcatgggcaaatat tatacgcaag gcgacaaggt gctgatgccg 1320 ctggcgattc aggttcatcatgccgtctgt gatggcttcc atgtcggcag aatgcttaat 1380 gaattacaac agtactgcgatgagtggcag ggcggggcgt aattttttta aggcagttat 1440 tggtgccctt aaacgcctggggtaatgact ctctagcttg aggcatcaaa taaaacgaaa 1500 ggctcagtcg aaagactgggcctttcgttt tatctgttgt ttgtcggtga acgctctcct 1560 gagtaggaca aatccgccgctctagagctg cctcgcgcgt ttcggtgatg acggtgaaaa 1620 cctctgacac atgcagctcccggagacggt cacagcttgt ctgtaagcgg atgccgggag 1680 cagacaagcc cgtcagggcgcgtcagcggg tgttggcggg tgtcggggcg cagccatgac 1740 ccagtcacgt agcgatagcggagtgtatac tggcttaact atgcggcatc agagcagatt 1800 gtactgagag tgcaccatatgcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac 1860 cgcatcaggc gctcttccgcttcctcgctc actgactcgc tgcgctcggt ctgtcggctg 1920 cggcgagcgg tatcagctcactcaaaggcg gtaatacggt tatccacaga atcaggggat 1980 aacgcaggaa agaacatgtgagcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 2040 gcgttgctgg cgtttttccataggctccgc ccccctgacg agcatcacaa aaatcgacgc 2100 tcaagtcaga ggtggcgaaacccgacagga ctataaagat accaggcgtt tccccctgga 2160 agctccctcg tgcgctctcctgttccgacc ctgccgctta ccggatacct gtccgccttt 2220 ctcccttcgg gaagcgtggcgctttctcaa tgctcacgct gtaggtatct cagttcggtg 2280 taggtcgttc gctccaagctgggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 2340 gccttatccg gtaactatcgtcttgagtcc aacccggtaa gacacgactt atcgccactg 2400 gcagcagcca ctggtaacaggattagcaga gcgaggtatg taggcggtgc tacagagttc 2460 ttgaagtggt ggcctaactacggctacact agaaggacag tatttggtat ctgcgctctg 2520 ctgaagccag ttaccttcggaaaaagagtt ggtagctctt gatccggcaa acaaaccacc 2580 gctggtagcg gtggtttttttgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 2640 caagaagatc ctttgatcttttctacgggg tctgacgctc agtggaacga aaactcacgt 2700 taagggattt tggtcatgagattatcaaaa aggatcttca cctagatcct tttaaattaa 2760 aaatgaagtt ttaaatcaatctaaagtata tatgagtaaa cttggtctga cagttaccaa 2820 tgcttaatca gtgaggcacctatctcagcg atctgtctat ttcgttcatc catagctgcc 2880 tgactccccg tcgtgtagataactacgata cgggagggct taccatctgg ccccagtgct 2940 gcaatgatac cgcgagacccacgctcaccg gctccagatt tatcagcaat aaaccagcca 3000 gccggaaggg ccgagcgcagaagtggtcct gcaactttat ccgcctccat ccagtctatt 3060 aattgttgcc gggaagctagagtaagtagt tcgccagtta atagtttgcg caacgttgtt 3120 gccattgcta caggcatcgtggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 3180 ggttcccaac gatcaaggcgagttacatga tcccccatgt tgtgcaaaaa agcggttagc 3240 tccttcggtc ctccgatcgttgtcagaagt aagttggccg cagtgttatc actcatggtt 3300 atggcagcac tgcataattctcttactgtc atgccatccg taagatgctt ttctgtgact 3360 ggtgagtact caaccaagtcattctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 3420 ccggcgtcaa tacgggataataccgcgcca catagcagaa ctttaaaagt gctcatcatt 3480 ggaaaacgtt cttcggggcgaaaactctca aggatcttac cgctgttgag atccagttcg 3540 atgtaaccca ctcgtgcacccaactgatct tcagcatctt ttactttcac cagcgtttct 3600 gggtgagcaa aaacaggaaggcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 3660 tgttgaatac tcatactcttcctttttcaa tattattgaa gcatttatca gggttattgt 3720 ctcatgagcg gatacatatttgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 3780 acatttcccc gaaaagtgccacctgacgtc taagaaacca ttattatcat gacattaacc 3840 tataaaaata ggcgtatcacgaggcccttt cgtcttcac 3879 26 3879 DNA Artificial sequencepNCO-AA-BglII-LuSy Expression vector 26 ctcgagaaat cataaaaaat ttatttgctttgtgagcgga taacaattat aatagattca 60 attgtgagcg gataacaatt tcacacagaattcattaaag aggagaaatt aactatgcag 120 atctacgaag gtaaactaac tgctgaaggccttcgtttcg gtatcgtagc atcacgtttt 180 aatcatgctc ttgtcgaccg tctggtggagggtgcaattg attgcatagt ccgtcatggc 240 ggccgtgaag aagacattac tctggttcgtgttccaggct catgggaaat accggttgct 300 gcgggtgaac tggcgcgtaa agaggacattgatgctgtta tcgcaattgg cgttctcatc 360 agaggcgcaa cgccacattt cgattatatcgcctctgaag tttcaaaagg cctcgcgaac 420 ctttcattag aactacgtaa acctatcaccttcggtgtta ttacagctga caccttggaa 480 caggctatcg agcgcgccgg cacaaaacacggcaacaaag gttgggaagc agcgctttct 540 gccattgaaa tggcaaactt attcaagtctctccgataag gatccgtcga cctgcagcca 600 agcttaatta gctgagcttg gactcctgttgatagatcca gtaatgacct cagaactcca 660 tctggatttg ttcagaacgc tcggttgccgccgggcgttt tttattggtg agaatccaag 720 ctagcttggc gagattttca ggagctaaggaagctaaaat ggagaaaaaa atcactggat 780 ataccaccgt tgatatatcc caatggcatcgtaaagaaca ttttgaggca tttcagtcag 840 ttgctcaatg tacctataac cagaccgttcagctggatat tacggccttt ttaaagaccg 900 taaagaaaaa taagcacaag ttttatccggcctttattca cattcttgcc cgcctgatga 960 atgctcatcc ggaatttcgt atggcaatgaaagacggtga gctggtgata tgggatagtg 1020 ttcacccttg ttacaccgtt ttccatgagcaaactgaaac gttttcatcg ctctggagtg 1080 aataccacga cgatttccgg cagtttctacacatatattc gcaagatgtg gcgtgttacg 1140 gtgaaaacct ggcctatttc cctaaagggtttattgagaa tatgtttttc gtctcagcca 1200 atccctgggt gagtttcacc agttttgatttaaacgtggc caatatggac aacttcttcg 1260 cccccgtttt caccatgcat gggcaaatattatacgcaag gcgacaaggt gctgatgccg 1320 ctggcgattc aggttcatca tgccgtctgtgatggcttcc atgtcggcag aatgcttaat 1380 gaattacaac agtactgcga tgagtggcagggcggggcgt aattttttta aggcagttat 1440 tggtgccctt aaacgcctgg ggtaatgactctctagcttg aggcatcaaa taaaacgaaa 1500 ggctcagtcg aaagactggg cctttcgttttatctgttgt ttgtcggtga acgctctcct 1560 gagtaggaca aatccgccgc tctagagctgcctcgcgcgt ttcggtgatg acggtgaaaa 1620 cctctgacac atgcagctcc cggagacggtcacagcttgt ctgtaagcgg atgccgggag 1680 cagacaagcc cgtcagggcg cgtcagcgggtgttggcggg tgtcggggcg cagccatgac 1740 ccagtcacgt agcgatagcg gagtgtatactggcttaact atgcggcatc agagcagatt 1800 gtactgagag tgcaccatat gcggtgtgaaataccgcaca gatgcgtaag gagaaaatac 1860 cgcatcaggc gctcttccgc ttcctcgctcactgactcgc tgcgctcggt ctgtcggctg 1920 cggcgagcgg tatcagctca ctcaaaggcggtaatacggt tatccacaga atcaggggat 1980 aacgcaggaa agaacatgtg agcaaaaggccagcaaaagg ccaggaaccg taaaaaggcc 2040 gcgttgctgg cgtttttcca taggctccgcccccctgacg agcatcacaa aaatcgacgc 2100 tcaagtcaga ggtggcgaaa cccgacaggactataaagat accaggcgtt tccccctgga 2160 agctccctcg tgcgctctcc tgttccgaccctgccgctta ccggatacct gtccgccttt 2220 ctcccttcgg gaagcgtggc gctttctcaatgctcacgct gtaggtatct cagttcggtg 2280 taggtcgttc gctccaagct gggctgtgtgcacgaacccc ccgttcagcc cgaccgctgc 2340 gccttatccg gtaactatcg tcttgagtccaacccggtaa gacacgactt atcgccactg 2400 gcagcagcca ctggtaacag gattagcagagcgaggtatg taggcggtgc tacagagttc 2460 ttgaagtggt ggcctaacta cggctacactagaaggacag tatttggtat ctgcgctctg 2520 ctgaagccag ttaccttcgg aaaaagagttggtagctctt gatccggcaa acaaaccacc 2580 gctggtagcg gtggtttttt tgtttgcaagcagcagatta cgcgcagaaa aaaaggatct 2640 caagaagatc ctttgatctt ttctacggggtctgacgctc agtggaacga aaactcacgt 2700 taagggattt tggtcatgag attatcaaaaaggatcttca cctagatcct tttaaattaa 2760 aaatgaagtt ttaaatcaat ctaaagtatatatgagtaaa cttggtctga cagttaccaa 2820 tgcttaatca gtgaggcacc tatctcagcgatctgtctat ttcgttcatc catagctgcc 2880 tgactccccg tcgtgtagat aactacgatacgggagggct taccatctgg ccccagtgct 2940 gcaatgatac cgcgagaccc acgctcaccggctccagatt tatcagcaat aaaccagcca 3000 gccggaaggg ccgagcgcag aagtggtcctgcaactttat ccgcctccat ccagtctatt 3060 aattgttgcc gggaagctag agtaagtagttcgccagtta atagtttgcg caacgttgtt 3120 gccattgcta caggcatcgt ggtgtcacgctcgtcgtttg gtatggcttc attcagctcc 3180 ggttcccaac gatcaaggcg agttacatgatcccccatgt tgtgcaaaaa agcggttagc 3240 tccttcggtc ctccgatcgt tgtcagaagtaagttggccg cagtgttatc actcatggtt 3300 atggcagcac tgcataattc tcttactgtcatgccatccg taagatgctt ttctgtgact 3360 ggtgagtact caaccaagtc attctgagaatagtgtatgc ggcgaccgag ttgctcttgc 3420 ccggcgtcaa tacgggataa taccgcgccacatagcagaa ctttaaaagt gctcatcatt 3480 ggaaaacgtt cttcggggcg aaaactctcaaggatcttac cgctgttgag atccagttcg 3540 atgtaaccca ctcgtgcacc caactgatcttcagcatctt ttactttcac cagcgtttct 3600 gggtgagcaa aaacaggaag gcaaaatgccgcaaaaaagg gaataagggc gacacggaaa 3660 tgttgaatac tcatactctt cctttttcaatattattgaa gcatttatca gggttattgt 3720 ctcatgagcg gatacatatt tgaatgtatttagaaaaata aacaaatagg ggttccgcgc 3780 acatttcccc gaaaagtgcc acctgacgtctaagaaacca ttattatcat gacattaacc 3840 tataaaaata ggcgtatcac gaggccctttcgtcttcac 3879 27 3876 DNA Artificial sequence pNCO-AA-LuSy-(BamHI)Expression vector 27 ctcgagaaat cataaaaaat ttatttgctt tgtgagcggataacaattat aatagattca 60 attgtgagcg gataacaatt tcacacagaa ttcattaaagaggagaaatt aactatgcaa 120 atctacgaag gtaaactaac tgctgaaggc cttcgtttcggtatcgtagc atcacgtttt 180 aatcatgctc ttgtcgaccg tctggtggag ggtgcaattgattgcatagt ccgtcatggc 240 ggccgtgaag aagacattac tctggttcgt gttccaggctcatgggaaat accggttgct 300 gcgggtgaac tggcgcgtaa agaggacatt gatgctgttatcgcaattgg cgttctcatc 360 agaggcgcaa cgccacattt cgattatatc gcctctgaagtttcaaaagg cctcgcgaac 420 ctttcattag aactacgtaa acctatcacc ttcggtgttattacagctga caccttggaa 480 caggctatcg agcgcgccgg cacaaaacac ggcaacaaaggttgggaagc agcgctttct 540 gccattgaaa tggcaaactt attcaagtct ctccgaggatccgtcgacct gcagccaagc 600 ttaattagct gagcttggac tcctgttgat agatccagtaatgacctcag aactccatct 660 ggatttgttc agaacgctcg gttgccgccg ggcgttttttattggtgaga atccaagcta 720 gcttggcgag attttcagga gctaaggaag ctaaaatggagaaaaaaatc actggatata 780 ccaccgttga tatatcccaa tggcatcgta aagaacattttgaggcattt cagtcagttg 840 ctcaatgtac ctataaccag accgttcagc tggatattacggccttttta aagaccgtaa 900 agaaaaataa gcacaagttt tatccggcct ttattcacattcttgcccgc ctgatgaatg 960 ctcatccgga atttcgtatg gcaatgaaag acggtgagctggtgatatgg gatagtgttc 1020 acccttgtta caccgttttc catgagcaaa ctgaaacgttttcatcgctc tggagtgaat 1080 accacgacga tttccggcag tttctacaca tatattcgcaagatgtggcg tgttacggtg 1140 aaaacctggc ctatttccct aaagggttta ttgagaatatgtttttcgtc tcagccaatc 1200 cctgggtgag tttcaccagt tttgatttaa acgtggccaatatggacaac ttcttcgccc 1260 ccgttttcac catgcatggg caaatattat acgcaaggcgacaaggtgct gatgccgctg 1320 gcgattcagg ttcatcatgc cgtctgtgat ggcttccatgtcggcagaat gcttaatgaa 1380 ttacaacagt actgcgatga gtggcagggc ggggcgtaatttttttaagg cagttattgg 1440 tgcccttaaa cgcctggggt aatgactctc tagcttgaggcatcaaataa aacgaaaggc 1500 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttgtcggtgaacg ctctcctgag 1560 taggacaaat ccgccgctct agagctgcct cgcgcgtttcggtgatgacg gtgaaaacct 1620 ctgacacatg cagctcccgg agacggtcac agcttgtctgtaagcggatg ccgggagcag 1680 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgtcggggcgcag ccatgaccca 1740 gtcacgtagc gatagcggag tgtatactgg cttaactatgcggcatcaga gcagattgta 1800 ctgagagtgc accatatgcg gtgtgaaata ccgcacagatgcgtaaggag aaaataccgc 1860 atcaggcgct cttccgcttc ctcgctcact gactcgctgcgctcggtctg tcggctgcgg 1920 cgagcggtat cagctcactc aaaggcggta atacggttatccacagaatc aggggataac 1980 gcaggaaaga acatgtgagc aaaaggccag caaaaggccaggaaccgtaa aaaggccgcg 2040 ttgctggcgt ttttccatag gctccgcccc cctgacgagcatcacaaaaa tcgacgctca 2100 agtcagaggt ggcgaaaccc gacaggacta taaagataccaggcgtttcc ccctggaagc 2160 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccggatacctgtc cgcctttctc 2220 ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgtaggtatctcag ttcggtgtag 2280 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccgttcagcccga ccgctgcgcc 2340 ttatccggta actatcgtct tgagtccaac ccggtaagacacgacttatc gccactggca 2400 gcagccactg gtaacaggat tagcagagcg aggtatgtaggcggtgctac agagttcttg 2460 aagtggtggc ctaactacgg ctacactaga aggacagtatttggtatctg cgctctgctg 2520 aagccagtta ccttcggaaa aagagttggt agctcttgatccggcaaaca aaccaccgct 2580 ggtagcggtg gtttttttgt ttgcaagcag cagattacgcgcagaaaaaa aggatctcaa 2640 gaagatcctt tgatcttttc tacggggtct gacgctcagtggaacgaaaa ctcacgttaa 2700 gggattttgg tcatgagatt atcaaaaagg atcttcacctagatcctttt aaattaaaaa 2760 tgaagtttta aatcaatcta aagtatatat gagtaaacttggtctgacag ttaccaatgc 2820 ttaatcagtg aggcacctat ctcagcgatc tgtctatttcgttcatccat agctgcctga 2880 ctccccgtcg tgtagataac tacgatacgg gagggcttaccatctggccc cagtgctgca 2940 atgataccgc gagacccacg ctcaccggct ccagatttatcagcaataaa ccagccagcc 3000 ggaagggccg agcgcagaag tggtcctgca actttatccgcctccatcca gtctattaat 3060 tgttgccggg aagctagagt aagtagttcg ccagttaatagtttgcgcaa cgttgttgcc 3120 attgctacag gcatcgtggt gtcacgctcg tcgtttggtatggcttcatt cagctccggt 3180 tcccaacgat caaggcgagt tacatgatcc cccatgttgtgcaaaaaagc ggttagctcc 3240 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcagtgttatcact catggttatg 3300 gcagcactgc ataattctct tactgtcatg ccatccgtaagatgcttttc tgtgactggt 3360 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggcgaccgagttg ctcttgcccg 3420 gcgtcaatac gggataatac cgcgccacat agcagaactttaaaagtgct catcattgga 3480 aaacgttctt cggggcgaaa actctcaagg atcttaccgctgttgagatc cagttcgatg 3540 taacccactc gtgcacccaa ctgatcttca gcatcttttactttcaccag cgtttctggg 3600 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaataagggcgac acggaaatgt 3660 tgaatactca tactcttcct ttttcaatat tattgaagcatttatcaggg ttattgtctc 3720 atgagcggat acatatttga atgtatttag aaaaataaacaaataggggt tccgcgcaca 3780 tttccccgaa aagtgccacc tgacgtctaa gaaaccattattatcatgac attaacctat 3840 aaaaataggc gtatcacgag gccctttcgt cttcac 387628 3876 DNA Artificial sequence pNCO-AA-BglII-LuSy-(BamHI) Expressionvector 28 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattataatagattca 60 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaattaactatgcag 120 atctacgaag gtaaactaac tgctgaaggc cttcgtttcg gtatcgtagcatcacgtttt 180 aatcatgctc ttgtcgaccg tctggtggag ggtgcaattg attgcatagtccgtcatggc 240 ggccgtgaag aagacattac tctggttcgt gttccaggct catgggaaataccggttgct 300 gcgggtgaac tggcgcgtaa agaggacatt gatgctgtta tcgcaattggcgttctcatc 360 agaggcgcaa cgccacattt cgattatatc gcctctgaag tttcaaaaggcctcgcgaac 420 ctttcattag aactacgtaa acctatcacc ttcggtgtta ttacagctgacaccttggaa 480 caggctatcg agcgcgccgg cacaaaacac ggcaacaaag gttgggaagcagcgctttct 540 gccattgaaa tggcaaactt attcaagtct ctccgaggat ccgtcgacctgcagccaagc 600 ttaattagct gagcttggac tcctgttgat agatccagta atgacctcagaactccatct 660 ggatttgttc agaacgctcg gttgccgccg ggcgtttttt attggtgagaatccaagcta 720 gcttggcgag attttcagga gctaaggaag ctaaaatgga gaaaaaaatcactggatata 780 ccaccgttga tatatcccaa tggcatcgta aagaacattt tgaggcatttcagtcagttg 840 ctcaatgtac ctataaccag accgttcagc tggatattac ggcctttttaaagaccgtaa 900 agaaaaataa gcacaagttt tatccggcct ttattcacat tcttgcccgcctgatgaatg 960 ctcatccgga atttcgtatg gcaatgaaag acggtgagct ggtgatatgggatagtgttc 1020 acccttgtta caccgttttc catgagcaaa ctgaaacgtt ttcatcgctctggagtgaat 1080 accacgacga tttccggcag tttctacaca tatattcgca agatgtggcgtgttacggtg 1140 aaaacctggc ctatttccct aaagggttta ttgagaatat gtttttcgtctcagccaatc 1200 cctgggtgag tttcaccagt tttgatttaa acgtggccaa tatggacaacttcttcgccc 1260 ccgttttcac catgcatggg caaatattat acgcaaggcg acaaggtgctgatgccgctg 1320 gcgattcagg ttcatcatgc cgtctgtgat ggcttccatg tcggcagaatgcttaatgaa 1380 ttacaacagt actgcgatga gtggcagggc ggggcgtaat ttttttaaggcagttattgg 1440 tgcccttaaa cgcctggggt aatgactctc tagcttgagg catcaaataaaacgaaaggc 1500 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacgctctcctgag 1560 taggacaaat ccgccgctct agagctgcct cgcgcgtttc ggtgatgacggtgaaaacct 1620 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatgccgggagcag 1680 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcagccatgaccca 1740 gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcagagcagattgta 1800 ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggagaaaataccgc 1860 atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtctgtcggctgcgg 1920 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatcaggggataac 1980 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaaaaaggccgcg 2040 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaatcgacgctca 2100 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttccccctggaagc 2160 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtccgcctttctc 2220 ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcagttcggtgtag 2280 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccgaccgctgcgcc 2340 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatcgccactggca 2400 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctacagagttcttg 2460 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctgcgctctgctg 2520 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaacaaaccaccgct 2580 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaaaggatctcaa 2640 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaactcacgttaa 2700 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatccttttaaattaaaaa 2760 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacagttaccaatgc 2820 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccatagctgcctga 2880 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccccagtgctgca 2940 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaaccagccagcc 3000 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatccagtctattaat 3060 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaacgttgttgcc 3120 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcattcagctccggt 3180 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagcggttagctcc 3240 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcactcatggttatg 3300 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttctgtgactggt 3360 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttgctcttgcccg 3420 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgctcatcattgga 3480 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatccagttcgatg 3540 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccagcgtttctggg 3600 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgacacggaaatgt 3660 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcagggttattgtctc 3720 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggttccgcgcaca 3780 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgacattaacctat 3840 aaaaataggc gtatcacgag gccctttcgt cttcac 3876 29 39 DNAArtificial sequence Synthetic oligonucleotide primer 29 gaggagaaattaaccatgaa tatcatacaa ggaaattta 39 30 44 DNA Artificial sequenceSynthetic oligonucleotide primer 30 tattatggat ccccatggtt attcgaaagaacggtttaag tttg 44 31 36 DNA Artificial sequence Syntheticoligonucleotide primer 31 ataatagaat tcattaaaga ggagaaatta accatg 36 3225 DNA Artificial sequence Synthetic oligonucleotide primer 32gtgagcggat aacaatttca cacag 25 33 36 DNA Artificial sequence Syntheticoligonucleotide primer 33 ataatagaat tcattaaaga ggagaaatta actatg 36 3426 DNA Artificial sequence Synthetic oligonucleotide primer 34gtataataga ttcaaattgt gagcgg 26 35 24 DNA Artificial sequence Syntheticoligonucleotide primer 35 agatattttc attaaagagg agaa 24 36 38 DNAArtificial sequence Synthetic oligonucleotide primer 36 tattatggatccttattcaa atgagcggtt taaatttg 38 37 29 DNA Artificial sequenceSynthetic oligonucleotide primer 37 gcagcttcat tcgaaacata atcgtaatg 2938 31 DNA Artificial sequence Synthetic oligonucleotide primer 38ggcagaaaca gctgaatcta cacctttgtt g 31 39 58 DNA Artificial sequenceSynthetic oligonucleotide primer 39 actatggcgg cggcgcgtag ctgcgcggccgctatgaata tcatacaagg aaatttag 58 40 41 DNA Artificial sequenceSynthetic oligonucleotide primer 40 tattatggat ccaaattatt caaatgagcggtttaaattt g 41 41 54 DNA Artificial sequence Synthetic oligonucleotideprimer 41 ataatagaat tcattaaaga ggagaaatta actatggcgg cggcgcgtag ctgc 5442 59 DNA Artificial sequence Synthetic oligonucleotide primer 42ttttcgggat ccttttaaac tgtttgcggc cgctaattca aatgagcggt ttaaatttg 59 4334 DNA Artificial sequence Synthetic oligonucleotide primer 43gaggagaaat taactatgat cagtctgatt gcgg 34 44 42 DNA Artificial sequenceSynthetic oligonucleotide primer 44 ctagccgtaa attctatagc ggccgcacgccgctccagaa tc 42 45 36 DNA Artificial sequence Synthetic oligonucleotideprimer 45 ataatacaat tgattaaaga ggagaaatta actatg 36 46 37 DNAArtificial sequence Synthetic oligonucleotide primer 46 gaggagaaattaactatgaa aatcgaagaa ggtaaac 37 47 30 DNA Artificial sequence Syntheticoligonucleotide primer 47 gcaggtcgac tctagcggcc gcgaattctg 30 48 56 DNAArtificial sequence Synthetic oligonucleotide primer 48 atagtggcgacaatgcggcc gctggtggag gcggaatgat cagtctgatt gcggcg 56 49 31 DNAArtificial sequence Synthetic oligonucleotide primer 49 ttctatggatccttaccgcc gctccagaat c 31 50 59 DNA Artificial sequence Syntheticoligonucleotide primer 50 ggtcagccgg ctgttcgtaa cgaacgtatg aatatcatacaaggaaattt agttggtac 59 51 71 DNA Artificial sequence Syntheticoligonucleotide primer 51 gaggagaaat taactatggg ggacggtgct gttcagccggacggtggtca gccggctgtt 60 cgtaacgaac g 71 52 54 DNA Artificial sequenceSynthetic oligonucleotide primer 52 ccaccgtccg gctgaacagc accgtcaccttcgaaagaac ggtttaagtt tgcc 54 53 65 DNA Artificial sequence Syntheticoligonucleotide primer 53 atatatggat cctaacgttc gttacgaaca gccggctgaccaccgtccgg ctgaacagca 60 ccgtc 65 54 63 DNA Artificial sequenceSynthetic oligonucleotide primer 54 catagcttcg aagatgccgc cgagtgcggccgcttcgaaa gaacggttta agtttgccat 60 ttc 63 55 60 DNA Artificial sequenceSynthetic oligonucleotide primer 55 tattatggat ccttagcgcc actccatcttcatagcttcg aagatgccgc cgagtgcggc 60 56 78 DNA Artificial sequenceSynthetic oligonucleotide primer 56 tattatggat ccttatttac cagagccaccaccagaacca ccgccacctt cgaaagaacg 60 gtttaagttt gccatttc 78 57 11 PRTArtificial sequence Synthetic peptide sequence 57 Gly Gly Gly Gly SerGly Gly Gly Ser Gly Lys 1 5 10 58 84 DNA Artificial sequence Syntheticoligonucleotide primer 58 tattatggat ccttagcagc caccaccaga gccaccaccagaaccaccgc caccttcgaa 60 agaacggttt aagtttgcca tttc 84 59 13 PRTArtificial sequence Synthetic peptide sequence 59 Gly Gly Gly Gly SerGly Gly Gly Ser Gly Gly Gly Cys 1 5 10 60 40 DNA Artificial sequenceSynthetic oligonucleotide primer 60 ataataataa agcttatgaa tatcatacaaggaaatttag 40 61 34 DNA Artificial sequence Synthetic oligonucleotideprimer 61 tattatgaat tcttattcga aagaacggtt taag 34 62 34 DNA Artificialsequence Synthetic oligonucleotide primer 62 gtggtgatgg tgatgttcgaaagaacggtt taag 34 63 33 DNA Artificial sequence Syntheticoligonucleotide primer 63 tattatggat ccttaatggt ggtgatggtg atg 33 64 63DNA Artificial sequence Synthetic oligonucleotide primer 64 gctgcgggtgaactggcgcg taaagaggac attgatgctg ttatcgcaat tggcgttctc 60 atc 63 65 63DNA Artificial sequence Synthetic oligonucleotide primer 65 ctaatgaaaggttcgcgagg ccttttgaaa cttcagaggc gatataatcg aaatgtggcg 60 ttg 63 66 64DNA Artificial sequence Synthetic oligonucleotide primer 66 actctggttcgtgttccagg ctcatgggaa ataccggttg ctgcgggtga actggcgcgt 60 aaag 64 67 70DNA Artificial sequence Synthetic oligonucleotide primer 67 ccaaggtgtcagctgtaata acaccgaagg tgataggttt acgtagttct aatgaaaggt 60 tcgcgaggcc 7068 73 DNA Artificial sequence Synthetic oligonucleotide primer 68ggagggtgca attgattgca tagtccgtca tggcggccgt gaagaagaca ttactctggt 60tcgtgttcca ggc 73 69 60 DNA Artificial sequence Syntheticoligonucleotide primer 69 gttgccgtgt tttgtgccgg cgcgctcgat agcctgttccaaggtgtcag ctgtaataac 60 70 71 DNA Artificial sequence Syntheticoligonucleotide primer 70 cggtatcgta gcatcacgtt ttaatcatgc tcttgtcgaccgtctggtgg agggtgcaat 60 tgattgcata g 71 71 69 DNA Artificial sequenceSynthetic oligonucleotide primer 71 gaataagttt gccatttcaa tggcagaaagcgctgcttcc caacctttgt tgccgtgttt 60 tgtgccggc 69 72 70 DNA Artificialsequence Synthetic oligonucleotide primer 72 atgcaaatct acgaaggtaaactaactgct gaaggccttc gtttcggtat cgtagcatca 60 cgttttaatc 70 73 50 DNAArtificial sequence Synthetic oligonucleotide primer 73 tattatggatccttatcgga gagacttgaa taagtttgcc atttcaatgg 50 74 59 DNA Artificialsequence Synthetic oligonucleotide primer 74 ataatagaat tcattaaagaggagaaatta actatgcaaa tctacgaagg taaactaac 59 75 37 DNA Artificialsequence Synthetic oligonucleotide primer 75 tattattata gcggccgctcggagagactt gaataag 37 76 60 DNA Artificial sequence Syntheticoligonucleotide primer 76 tattattata gcggccgcat ggtggtgatg gtgatgtcggagagacttga ataagtttgc 60 77 72 DNA Artificial sequence Syntheticoligonucleotide primer 77 tattattata gcggccgcgc cagaaccgcc atggtggtgatggtgatgtc ggagagactt 60 gaataagttt gc 72 78 10 PRT Artificial sequenceSynthetic peptide sequence 78 His His His His His His Gly Gly Ser Gly 15 10 79 30 DNA Artificial sequence Synthetic oligonucleotide primer 79tattattata accggtattt caaatgcgcc 30 80 50 DNA Artificial sequenceSynthetic oligonucleotide primer 80 ataatagaat tcattaaaga ggagaaattaactatgcaga tctacgaagg 50 81 36 DNA Artificial sequence Syntheticoligonucleotide primer 81 tattatggat cctcggagag acttgaataa gtttgc 36 8224 PRT Artificial sequence Linker peptide sequence 82 Ser Asn Asn AsnAsn Asn Asn Asn Asn Asn Asn Leu Gly Ile Glu Gly 1 5 10 15 Arg Ile SerGlu Phe Ala Ala Ala 20 83 8 PRT Artificial sequence Linker peptidesequence 83 Leu Ala Ala Ala Gly Gly Gly Gly 1 5 84 11 PRT Artificialsequence Linker peptide sequence 84 Gly Ser Val Asp Leu Gln Pro Ser LeuIle Ser 1 5 10 85 13 PRT Artificial sequence Biotinylation peptidesequence 85 Leu Gly Gly Ile Phe Glu Ala Met Lys Met Glu Trp Arg 1 5 1086 6 PRT Artificial sequence Linker peptide sequence 86 His His His AlaAla Ala 1 5 87 13 PRT Artificial sequence Linker peptide sequence 87 HisHis His His His His Gly Gly Ser Gly Ala Ala Ala 1 5 10 88 19 PRTArtificial sequence Mink enteritis virus VP2 antigenically peptide 88Met Gly Asp Gly Ala Val Gln Pro Asp Gly Gly Gln Pro Ala Val Arg 1 5 1015 Asn Glu Arg 89 18 PRT Artificial sequence Mink enteritis virus VP2antigenically active peptide 89 Gly Asp Gly Ala Val Gln Pro Asp Gly GlyGln Pro Ala Val Arg Asn 1 5 10 15 Glu Arg 90 9 PRT Artificial sequenceFLAG peptide sequence 90 Met Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 91 6PRT Artificial sequence His6 peptide sequence 91 His His His His His His1 5 92 10 PRT Artificial sequence Linker peptide sequence 92 Gly Gly GlyGly Ser Gly Gly Gly Ser Gly 1 5 10 93 11 PRT Artificial sequence Linkerpeptide sequence 93 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 1 5 1094 160 PRT Mycobacterium avium 94 Met Ser Pro Ala Ala Gly Val Pro GluMet Pro Ala Leu Asp Ala Ser 1 5 10 15 Gly Val Arg Leu Gly Ile Val AlaSer Thr Trp His Ser Arg Ile Cys 20 25 30 Asp Ala Leu Leu Ala Gly Ala ArgLys Val Ala Ala Asp Ser Gly Val 35 40 45 Glu Asn Pro Thr Val Val Arg ValLeu Gly Ala Ile Glu Ile Pro Val 50 55 60 Val Ala Gln Glu Leu Ala Arg AsnHis Asp Ala Val Val Ala Leu Gly 65 70 75 80 Val Val Ile Arg Gly Gln ThrPro His Phe Glu Tyr Val Cys Asp Ala 85 90 95 Val Thr Gln Gly Ile Thr ArgVal Ser Leu Asp Ala Ser Thr Pro Val 100 105 110 Ala Asn Gly Val Leu ThrThr Asp Asn Glu Gln Gln Ala Leu Asp Arg 115 120 125 Ala Gly Leu Pro AspSer Ala Glu Asp Lys Gly Ala Gln Ala Ala Gly 130 135 140 Ala Ala Leu SerAla Ala Leu Thr Leu Arg Glu Leu Arg Ala Arg Ser 145 150 155 160 95 154PRT Mycobacterium tuberculosis 95 Met Pro Asp Leu Pro Ser Leu Asp AlaSer Gly Val Arg Leu Ala Ile 1 5 10 15 Val Ala Ser Ser Trp His Gly LysIle Cys Asp Ala Leu Leu Asp Gly 20 25 30 Ala Arg Lys Val Ala Ala Gly CysGly Leu Asp Asp Pro Thr Val Val 35 40 45 Arg Val Leu Gly Ala Ile Glu IlePro Val Val Ala Gln Glu Leu Ala 50 55 60 Arg Asn His Asp Ala Val Val AlaLeu Gly Val Val Ile Arg Gly Gln 65 70 75 80 Thr Pro His Phe Asp Tyr ValCys Asp Ala Val Thr Gln Gly Leu Thr 85 90 95 Arg Val Ser Leu Asp Ser SerThr Pro Ile Ala Asn Gly Val Leu Thr 100 105 110 Thr Asn Thr Glu Glu GlnAla Leu Asp Arg Ala Gly Leu Pro Thr Ser 115 120 125 Ala Glu Asp Lys GlyAla Gln Ala Thr Val Ala Ala Leu Ala Thr Ala 130 135 140 Leu Thr Leu ArgGlu Leu Arg Ala His Ser 145 150 96 163 PRT Corynebacterium ammoniagenes96 Met Ser Lys Glu Gly Leu Pro Glu Val Ala Thr Ile Asp Ala Thr Gly 1 510 15 Ile Ser Val Ala Val Ile Ser Ala Thr Trp Asn Ala Asp Ile Cys Asp 2025 30 Arg Leu His Glu Arg Ala Leu Ala His Ala Gln Gln Leu Gly Ala Glu 3540 45 Ala Asp Gly Phe Arg Val Val Gly Ala Leu Glu Ile Pro Val Ala Val 5055 60 Gln Glu Ala Ala Arg His Tyr Asp Ala Val Val Ala Leu Gly Cys Val 6570 75 80 Ile Arg Gly Gly Thr Pro His Phe Asp Tyr Val Cys Asp Ser Val Thr85 90 95 Gln Gly Leu Thr Arg Ile Ala Leu Asp Thr Ser Lys Pro Ile Ala Asn100 105 110 Gly Val Leu Thr Val Asn Thr His Asp Gln Ala Val Asp Arg SerGly 115 120 125 Ala Pro Gly Ala Ala Glu Asp Lys Gly Val Glu Ala Met GlnAla Ala 130 135 140 Leu Asp Thr Val Leu Gln Leu Arg Asn Ile Lys Glu ArgAla Ser Lys 145 150 155 160 Arg Gly Leu 97 155 PRT Chlorobium tepidum 97Met Gln Val Gln Asn Ile Glu Gly Ser Leu Asn Ala Ser Gly Leu Lys 1 5 1015 Phe Ala Leu Val Val Ser Arg Phe Asn Asp Phe Ile Gly Gln Lys Leu 20 2530 Val Glu Gly Ala Ile Asp Cys Ile Val Arg His Gly Gly Ser Ala Asp 35 4045 Glu Ile Thr Val Ile Arg Cys Pro Gly Ala Phe Glu Leu Pro Ser Val 50 5560 Thr Arg Lys Ala Met Leu Ser Gly Lys Tyr Asp Ala Ile Val Thr Leu 65 7075 80 Gly Val Ile Ile Arg Gly Ser Thr Pro His Phe Asp Val Ile Ala Ala 8590 95 Glu Ala Thr Lys Gly Ile Ala Gln Val Gly Met Glu Ala Ala Ile Pro100 105 110 Val Ser Phe Gly Val Leu Thr Thr Glu Asn Leu Glu Gln Ala IleGlu 115 120 125 Arg Ala Gly Thr Lys Ala Gly Asn Lys Gly Phe Asp Ala AlaLeu Ala 130 135 140 Ala Ile Glu Met Ala Asn Leu Tyr Lys Gln Leu 145 150155 98 154 PRT Aquifex aeolicus 98 Met Gln Ile Tyr Glu Gly Lys Leu ThrAla Glu Gly Leu Arg Phe Gly 1 5 10 15 Ile Val Ala Ser Arg Phe Asn HisAla Leu Val Asp Arg Leu Val Glu 20 25 30 Gly Ala Ile Asp Cys Ile Val ArgHis Gly Gly Arg Glu Glu Asp Ile 35 40 45 Thr Leu Val Arg Val Pro Gly SerTrp Glu Ile Pro Val Ala Ala Gly 50 55 60 Glu Leu Ala Arg Lys Glu Asp IleAsp Ala Val Ile Ala Ile Gly Val 65 70 75 80 Leu Ile Arg Gly Ala Thr ProHis Phe Asp Tyr Ile Ala Ser Glu Val 85 90 95 Ser Lys Gly Leu Ala Asn LeuSer Leu Glu Leu Arg Lys Pro Ile Thr 100 105 110 Phe Gly Val Ile Thr AlaAsp Thr Leu Glu Gln Ala Ile Glu Arg Ala 115 120 125 Gly Thr Lys His GlyAsn Lys Gly Trp Glu Ala Ala Leu Ser Ala Ile 130 135 140 Glu Met Ala AsnLeu Phe Lys Ser Leu Arg 145 150 99 165 PRT Thermotoga maritima 99 MetLys Val Val Gln Gly Asp Tyr Arg Gly Glu Gly Leu Lys Ile Ala 1 5 10 15Val Val Val Pro Arg Phe Asn Asp Leu Val Thr Ser Lys Leu Leu Glu 20 25 30Gly Ala Leu Asp Gly Leu Lys Arg His Gly Val Ser Asp Glu Asn Ile 35 40 45Thr Val Val Arg Ile Pro Gly Ser Met Glu Ala Ile Tyr Thr Leu Lys 50 55 60Arg Leu Leu Asp Leu Gly Val His Asp Ala Ile Ile Val Leu Gly Ala 65 70 7580 Val Ile Arg Gly Glu Thr Tyr His Phe Asn Val Val Ala Asn Glu Ile 85 9095 Gly Lys Ala Val Ala Gln Phe Asn Met Thr Ser Asp Ile Pro Ile Val 100105 110 Phe Gly Val Leu Thr Thr Asp Thr Leu Glu Gln Ala Leu Asn Arg Ala115 120 125 Gly Ala Lys Ser Gly Asn Lys Gly Phe Glu Ala Ala Met Val AlaIle 130 135 140 Glu Met Ala Asn Leu Arg Lys Arg Leu Arg Arg Asp Val PheGlu Ser 145 150 155 160 Asp Ser Asn Gly Arg 165 100 154 PRT Bacillussubtilis 100 Met Asn Ile Ile Gln Gly Asn Leu Val Gly Thr Gly Leu Lys IleGly 1 5 10 15 Ile Val Val Gly Arg Phe Asn Asp Phe Ile Thr Ser Lys LeuLeu Ser 20 25 30 Gly Ala Glu Asp Ala Leu Leu Arg His Gly Val Asp Thr AsnAsp Ile 35 40 45 Asp Val Ala Trp Val Pro Gly Ala Phe Glu Ile Pro Phe AlaAla Lys 50 55 60 Lys Met Ala Glu Thr Lys Lys Tyr Asp Ala Ile Ile Thr LeuGly Thr 65 70 75 80 Val Ile Arg Gly Ala Thr Thr His Tyr Asp Tyr Val CysAsn Glu Ala 85 90 95 Ala Lys Gly Ile Ala Gln Ala Ala Asn Thr Thr Gly ValPro Val Ile 100 105 110 Phe Gly Ile Val Thr Thr Glu Asn Ile Glu Gln AlaIle Glu Arg Ala 115 120 125 Gly Thr Lys Ala Gly Asn Lys Gly Val Asp CysAla Val Ser Ala Ile 130 135 140 Glu Met Ala Asn Leu Asn Arg Ser Phe Glu145 150 101 154 PRT Bacillus amyloliquefaciens 101 Met Asn Ile Ile GlnGly Asn Leu Val Gly Thr Gly Leu Lys Ile Gly 1 5 10 15 Ile Val Val GlyArg Phe Asn Glu Phe Ile Thr Ser Lys Leu Leu Ser 20 25 30 Gly Ala Glu AspThr Leu Ile Arg His Gly Val Glu Ser Asn Asp Ile 35 40 45 Asp Val Ala TrpVal Pro Gly Ala Phe Glu Ile Pro Phe Ala Ala Lys 50 55 60 Lys Met Ala GluThr Lys Lys Tyr Asp Ala Val Ile Thr Leu Gly Thr 65 70 75 80 Val Ile ArgGly Ala Thr Thr His Tyr Asp Tyr Val Cys Asn Glu Ala 85 90 95 Ala Lys GlyIle Ala Gln Ala Gly Thr Ala Thr Gly Val Pro Val Ile 100 105 110 Phe GlyIle Val Thr Thr Glu Thr Ile Glu Gln Ala Ile Glu Arg Ala 115 120 125 GlyThr Lys Ala Gly Asn Lys Gly Ala Asp Cys Ala Val Ser Ala Ile 130 135 140Glu Met Ala Asn Leu Asn Arg Ser Phe Glu 145 150 102 153 PRTActinobacillus pleuropneumoniae 102 Met Ala Lys Ile Thr Gly Asn Leu ValAla Thr Gly Leu Lys Phe Gly 1 5 10 15 Ile Val Thr Ala Arg Phe Asn AspPhe Ile Asn Asp Lys Leu Leu Ser 20 25 30 Gly Ala Ile Asp Thr Leu Val ArgHis Gly Ala Tyr Glu Asn Asp Ile 35 40 45 Asp Thr Ala Trp Val Pro Gly AlaPhe Glu Ile Pro Leu Val Ala Lys 50 55 60 Lys Met Ala Asn Ser Gly Lys TyrAsp Ala Val Ile Cys Leu Gly Thr 65 70 75 80 Val Ile Arg Gly Ser Thr ThrHis Tyr Asp Tyr Val Cys Asn Glu Ala 85 90 95 Ala Lys Gly Ile Gly Ala ValAla Leu Glu Thr Gly Val Pro Val Ile 100 105 110 Phe Gly Val Leu Thr ThrGlu Asn Ile Glu Gln Ala Ile Glu Arg Ala 115 120 125 Gly Thr Lys Ala GlyAsn Lys Gly Ser Glu Cys Ala Leu Gly Ala Ile 130 135 140 Glu Ile Val AsnVal Leu Lys Ala Ile 145 150 103 155 PRT Streptococcus pneumoniae 103 MetAsn Thr Tyr Glu Gly Asn Leu Val Ala Asn Asn Ile Lys Ile Gly 1 5 10 15Ile Val Val Ala Arg Phe Asn Glu Phe Ile Thr Ser Lys Leu Leu Ser 20 25 30Gly Ala Leu Asp Asn Leu Lys Arg Glu Asn Val Asn Glu Lys Asp Ile 35 40 45Glu Val Ala Trp Val Pro Gly Ala Phe Glu Ile Pro Leu Ile Ala Ser 50 55 60Lys Met Ala Lys Ser Lys Lys Tyr Asp Ala Ile Ile Cys Leu Gly Ala 65 70 7580 Val Ile Arg Gly Asn Thr Ser His Tyr Asp Tyr Val Cys Ser Glu Val 85 9095 Ser Lys Gly Ile Ala Gln Ile Ser Leu Asn Ser Glu Ile Pro Val Met 100105 110 Phe Gly Val Leu Thr Thr Asp Thr Ile Glu Gln Ala Ile Glu Arg Ala115 120 125 Gly Thr Lys Ala Gly Asn Lys Gly Ser Glu Cys Ala Gln Gly AlaIle 130 135 140 Glu Met Val Asn Leu Ile Arg Thr Leu Asp Ala 145 150 155104 154 PRT Staphylococcus aureus 104 Met Asn Phe Glu Gly Lys Leu IleGly Lys Asp Leu Lys Val Ala Ile 1 5 10 15 Val Val Ser Arg Phe Asn AspPhe Ile Thr Gly Arg Leu Leu Glu Gly 20 25 30 Ala Lys Asp Thr Leu Ile ArgHis Asp Val Asn Glu Asp Asn Ile Asp 35 40 45 Val Ala Phe Val Pro Gly AlaPhe Glu Ile Pro Leu Val Ala Lys Lys 50 55 60 Leu Ala Ser Ser Gly Asn TyrAsp Ala Val Ile Thr Leu Gly Cys Val 65 70 75 80 Ile Arg Gly Ala Thr SerHis Tyr Asp Tyr Val Cys Asn Glu Val Ala 85 90 95 Lys Gly Val Ser Lys ValAsn Asp Gln Thr Asn Val Pro Val Ile Phe 100 105 110 Gly Ile Leu Thr ThrGlu Ser Ile Glu Gln Ala Val Glu Arg Ala Gly 115 120 125 Thr Lys Ala GlyAsn Lys Gly Ala Glu Ala Ala Val Ser Ala Ile Glu 130 135 140 Met Ala AsnLeu Leu Lys Ser Ile Lys Ala 145 150 105 156 PRT Vibrio cholerae 105 MetLys Val Ile Glu Gly Gly Phe Pro Ala Pro Asn Ala Lys Ile Ala 1 5 10 15Ile Val Ile Ser Arg Phe Asn Ser Phe Ile Asn Glu Ser Leu Leu Ser 20 25 30Gly Ala Ile Asp Thr Leu Lys Arg His Gly Gln Ile Ser Asp Asp Asn 35 40 45Ile Thr Val Val Arg Cys Pro Gly Ala Val Glu Leu Pro Leu Val Ala 50 55 60Gln Arg Val Ala Lys Thr Gly Asp Tyr Asp Ala Ile Val Ser Leu Gly 65 70 7580 Cys Val Ile Arg Gly Gly Thr Pro His Phe Asp Tyr Val Cys Ser Glu 85 9095 Met Asn Lys Gly Leu Ala Gln Val Ser Leu Glu Phe Ser Ile Pro Val 100105 110 Ala Phe Gly Val Leu Thr Val Asp Thr Ile Asp Gln Ala Ile Glu Arg115 120 125 Ala Gly Thr Lys Ala Gly Asn Lys Gly Ala Glu Ala Ala Leu SerAla 130 135 140 Leu Glu Met Ile Asn Val Leu Ser Glu Ile Asp Ser 145 150155 106 155 PRT Photobacterium phosporeum 106 Met Lys Val Ile Glu GlyAla Ile Val Ala Pro Asn Ala Lys Val Ala 1 5 10 15 Ile Val Ile Ala ArgPhe Asn Ser Phe Ile Asn Glu Ser Leu Leu Ser 20 25 30 Gly Ala Leu Asp ThrLeu Lys Arg Gln Gly Gln Val Ser Tyr Asp Asn 35 40 45 Ile Thr Ile Ile ArgCys Pro Gly Ala Tyr Glu Leu Pro Leu Val Ala 50 55 60 Gln Leu Thr Ala LysSer Asp Arg Tyr Asp Ala Ile Ile Ala Leu Gly 65 70 75 80 Ser Val Ile ArgGly Gly Thr His Phe Glu Tyr Val Ala Ser Glu Cys 85 90 95 Asn Lys Gly LeuAla Gln Val Ala Leu Asp Tyr Asn Ile Pro Val Ala 100 105 110 Phe Gly ValLeu Thr Val Asp Tyr Leu Glu Gln Ala Ile Glu Arg Ala 115 120 125 Gly ThrLys Ala Gly Asn Lys Gly Ala Glu Ala Ala Leu Met Leu Leu 130 135 140 GluMet Val Asn Ile Leu Ala Gln Val Glu Ser 145 150 155 107 158 PRTShewanella putrefaciens 107 Met Asn Val Val Gln Gly Asn Ile Glu Ala LysAsn Ala Lys Val Ala 1 5 10 15 Ile Val Ile Ser Arg Phe Asn Ser Phe LeuVal Glu Ser Leu Leu Glu 20 25 30 Gly Ala Leu Asp Thr Leu Lys Arg Phe GlyGln Val Ser Asp Glu Asn 35 40 45 Ile Thr Val Val Arg Val Pro Gly Ala ValGlu Leu Pro Leu Ala Ala 50 55 60 Arg Arg Val Ala Ala Ser Gly Lys Phe AspGly Ile Ile Ala Leu Gly 65 70 75 80 Ala Val Ile Arg Gly Gly Thr Pro HisPhe Asp Phe Val Ala Gly Glu 85 90 95 Cys Asn Lys Gly Leu Ala Gln Ile AlaLeu Glu Phe Asp Leu Pro Val 100 105 110 Ala Phe Gly Val Leu Thr Thr AspThr Ile Glu Gln Ala Ile Glu Arg 115 120 125 Ser Gly Thr Lys Ala Gly AsnLys Gly Gly Glu Ala Ala Leu Ser Leu 130 135 140 Leu Glu Met Val Asn ValLeu Gln Gln Leu Glu Gln Gln Leu 145 150 155 108 144 PRT Photobacteriumleiognathi 108 Met Lys Leu Leu Lys Gly Val Asp Cys Thr Ser Cys Cys IleAla Ile 1 5 10 15 Val Ile Ala Arg Phe Asn Ser Phe Ile Asn Glu Asn LeuLeu Ser Gly 20 25 30 Ala Ile Asn Ala Leu Gln Arg Lys Gly Gln Val Lys AlaGlu Asn Ile 35 40 45 Thr Val Ile Arg Cys Pro Gly Ala Tyr Glu Leu Pro LeuAla Ala Gln 50 55 60 Gln Ile Ala Lys Gln Gly Asn Tyr Asp Ala Ile Ile AlaIle Gly Ala 65 70 75 80 Val Ile Arg Gly Gly Thr Pro His Phe Asp Phe ValAla Gly Glu Cys 85 90 95 Asn Lys Gly Leu Ala Gln Val Ala Leu Glu Tyr GlnThr Pro Val Ala 100 105 110 Phe Gly Val Leu Thr Val Asp Ser Ile Glu GlnAla Ile Glu Arg Ala 115 120 125 Gly Thr Lys Met Gly Asn Lys Gly Glu GluAla Ala Leu Ser Ala Leu 130 135 140 109 156 PRT Shigella flexneri 109Met Asn Ile Ile Glu Ala Asn Val Ala Thr Pro Asp Ala Arg Val Ala 1 5 1015 Ile Thr Ile Ala Arg Phe Asn Asn Phe Ile Asn Asp Ser Leu Leu Glu 20 2530 Gly Ala Ile Asp Ala Leu Lys Arg Ile Gly Gln Val Lys Asp Glu Asn 35 4045 Ile Thr Val Val Trp Val Pro Gly Ala Tyr Glu Leu Pro Leu Ala Ala 50 5560 Gly Ala Leu Ala Lys Thr Gly Lys Tyr Asp Ala Val Ile Ala Leu Gly 65 7075 80 Thr Val Ile Arg Gly Gly Thr Ala His Phe Glu Tyr Val Ala Gly Gly 8590 95 Ala Ser Asn Gly Leu Ala His Val Ala Gln Asp Ser Glu Ile Pro Val100 105 110 Ala Phe Gly Val Leu Thr Thr Glu Ser Ile Glu Gln Ala Ile GluArg 115 120 125 Ala Gly Thr Lys Ala Gly Asn Lys Gly Ala Glu Ala Ala LeuThr Ala 130 135 140 Leu Glu Met Ile Asn Val Leu Lys Ala Ile Lys Ala 145150 155 110 156 PRT Escherichia coli 110 Met Asn Ile Ile Glu Ala Asn ValAla Thr Pro Asp Ala Arg Val Ala 1 5 10 15 Ile Thr Ile Ala Arg Phe AsnAsn Phe Ile Asn Asp Ser Leu Leu Glu 20 25 30 Gly Ala Ile Asp Ala Leu LysArg Ile Gly Gln Val Lys Asp Glu Asn 35 40 45 Ile Thr Val Val Trp Val ProGly Ala Tyr Glu Leu Pro Leu Ala Ala 50 55 60 Gly Ala Leu Ala Lys Thr GlyLys Tyr Asp Ala Val Ile Ala Leu Gly 65 70 75 80 Thr Val Ile Arg Gly GlyThr Ala His Phe Glu Tyr Val Ala Gly Gly 85 90 95 Ala Ser Asn Gly Leu AlaHis Val Ala Gln Asp Ser Glu Ile Pro Val 100 105 110 Ala Phe Gly Val LeuThr Thr Glu Ser Ile Glu Gln Ala Ile Glu Arg 115 120 125 Ala Gly Thr LysAla Gly Asn Lys Gly Ala Glu Ala Ala Leu Thr Ala 130 135 140 Leu Glu MetIle Asn Val Leu Lys Ala Ile Lys Ala 145 150 155 111 157 PRT Haemophilusinfluenzae 111 Met Lys Val Leu Glu Gly Ser Val Ala Ala Pro Asn Ala LysVal Ala 1 5 10 15 Val Val Ile Ala Arg Phe Asn Ser Phe Ile Asn Glu SerLeu Leu Glu 20 25 30 Gly Ala Ile Asp Ala Leu Lys Arg Ile Gly Gln Val LysAsp Glu Asn 35 40 45 Ile Thr Ile Val Arg Thr Pro Gly Ala Tyr Glu Leu ProLeu Val Ala 50 55 60 Arg Arg Leu Ala Glu Ser Lys Lys Phe Asp Ala Ile ValAla Leu Gly 65 70 75 80 Thr Val Ile Arg Gly Gly Thr Ala His Phe Glu TyrVal Ala Gly Glu 85 90 95 Ala Ser Ser Gly Leu Gly Lys Val Ala Met Asp AlaGlu Ile Pro Val 100 105 110 Ala Phe Gly Val Leu Thr Thr Glu Asn Ile GluGln Ala Ile Glu Arg 115 120 125 Ala Gly Thr Lys Ala Gly Asn Lys Gly AlaGlu Ala Ala Leu Thr Ala 130 135 140 Leu Glu Met Val Asn Leu Ile Gln GlnIle Asp Ala Ala 145 150 155 112 156 PRT Dehalospirillum multivorans 112Met Asn Ile Val Glu Gly Lys Leu Ser Leu Asn Gly Asp Glu Lys Val 1 5 1015 Ala Ile Ile Asn Ala Arg Phe Asn His Ile Ile Thr Asp Arg Leu Val 20 2530 Glu Gly Ala Arg Asp Ala Tyr Leu Arg His Gly Gly Lys Asp Glu Asn 35 4045 Leu Asp Leu Val Leu Val Pro Gly Ala Phe Glu Ile Pro Met Ala Leu 50 5560 Asn Arg Leu Leu Ala Cys Ser Lys Tyr Asp Ala Val Cys Cys Leu Gly 65 7075 80 Ala Val Ile Arg Gly Ser Thr Pro His Phe Asp Tyr Val Ser Ala Glu 8590 95 Val Thr Lys Gly Val Ala Asn Val Ala Leu Gln Phe Ala Lys Pro Val100 105 110 Ala Phe Gly Val Leu Thr Val Asp Ser Ile Glu Gln Ala Ile GluArg 115 120 125 Ala Gly Ser Lys Ala Gly Asn Lys Gly Phe Glu Ala Met ValThr Val 130 135 140 Ile Glu Leu Leu Ser Leu Tyr Ser Ala Leu Lys Asn 145150 155 113 156 PRT Helicobacter pylori 113 Met Gln Ile Ile Glu Gly LysLeu Gln Leu Gln Gly Asn Glu Arg Val 1 5 10 15 Ala Ile Leu Thr Ser ArgPhe Asn His Ile Ile Thr Asp Arg Leu Gln 20 25 30 Glu Gly Ala Met Asp CysPhe Lys Arg His Gly Gly Asp Glu Asp Leu 35 40 45 Leu Asp Ile Val Leu ValPro Gly Ala Tyr Glu Leu Pro Phe Ile Leu 50 55 60 Asp Lys Leu Leu Glu SerGlu Lys Tyr Asp Gly Val Cys Val Leu Gly 65 70 75 80 Ala Ile Ile Arg GlyGly Thr Pro His Phe Asp Tyr Val Ser Ala Glu 85 90 95 Ala Thr Lys Gly IleAla His Ala Met Leu Lys Tyr Ser Met Pro Val 100 105 110 Ser Phe Gly ValLeu Thr Thr Asp Asn Ile Glu Gln Ala Ile Glu Arg 115 120 125 Ala Gly SerLys Ala Gly Asn Lys Gly Phe Glu Ala Met Ser Thr Leu 130 135 140 Ile GluLeu Leu Ser Leu Cys Gln Thr Leu Lys Gly 145 150 155 114 155 PRTDeinococcus radiodurans 114 Met Gln Arg Ile Glu Ala Thr Leu Leu Ala HisAsp Leu Lys Phe Ala 1 5 10 15 Leu Val Ser Thr Arg Trp Asn His Leu IleVal Asp Arg Leu Val Glu 20 25 30 Gly Ala Glu Leu Ala Phe Val Gln His GlyGly Lys Thr Glu Asn Leu 35 40 45 Asp His Phe Leu Val Pro Gly Ser Tyr GluVal Pro Leu Val Ala Arg 50 55 60 Arg Leu Ala Glu Thr Gly Arg Tyr Asp AlaVal Val Cys Leu Gly Ala 65 70 75 80 Val Ile Lys Gly Asp Thr Asp His TyrAsp Phe Val Ala Gly Gly Ala 85 90 95 Ala Asn Gly Ile Leu Asn Thr Ser LeuHis Thr Gly Val Pro Val Ala 100 105 110 Phe Gly Val Leu Thr Thr Asp ThrVal Glu Gln Ala Leu Asn Arg Ala 115 120 125 Gly Ile Lys Ala Gly Asn LysGly Gly Glu Ala Val Leu Ala Met Ile 130 135 140 Glu Thr Ala Asn Leu LeuLys Gln Ile Glu Arg 145 150 155 115 164 PRT Synechocystis sp. 115 MetThr Val Tyr Glu Gly Ser Phe Thr Pro Pro Ala Arg Pro Phe Arg 1 5 10 15Phe Ala Leu Val Ile Ala Arg Phe Asn Asp Leu Val Thr Glu Lys Leu 20 25 30Leu Ser Gly Cys Gln Asp Cys Leu Lys Arg His Gly Ile Asp Val Asp 35 40 45Pro Ala Gly Thr Gln Val Asp Tyr Ile Trp Val Pro Gly Ser Phe Glu 50 55 60Val Pro Leu Val Thr Arg Lys Leu Ala Val Ser Gly Gln Tyr Asp Ala 65 70 7580 Ile Ile Cys Leu Gly Ala Val Ile Arg Gly Gln Thr Pro His Phe Asp 85 9095 Phe Val Ala Gly Glu Ala Ala Lys Gly Ile Ala Ala Ile Ala Ser Gln 100105 110 Thr Gly Val Pro Val Ile Phe Gly Ile Leu Thr Thr Asp Thr Met Gln115 120 125 Gln Ala Leu Glu Arg Ala Gly Ile Lys Ser Asn His Gly Trp GlyTyr 130 135 140 Ala Met Asn Ala Leu Glu Met Ala Ser Leu Met Arg Ala MetAla Pro 145 150 155 160 Leu Thr Glu Gly 116 161 PRT Porphyromonasgingivalis 116 Met Ala Thr Ala Tyr His Asn Leu Ser Asp Tyr Asp Tyr GluSer Val 1 5 10 15 Pro Cys Gly Lys Asp Leu Arg Ile Gly Ile Ala Val AlaGlu Trp Asn 20 25 30 His Asn Ile Thr Glu Pro Leu Met Lys Gly Ala Ile AspThr Leu Leu 35 40 45 Glu His Gly Val Ser Ala Asp Asn Ile Ile Val Gln HisVal Pro Gly 50 55 60 Thr Phe Glu Leu Thr Tyr Ala Ser Ala Tyr Leu Ala GluGln His Glu 65 70 75 80 Val Asp Ala Val Ile Ala Ile Gly Cys Val Val ArgGly Asp Thr Pro 85 90 95 His Phe Asp Tyr Ile Cys Gln Gly Val Thr Gln GlyIle Thr Gln Leu 100 105 110 Asn Ile Asp Gly Phe Val Pro Val Ile Phe GlyVal Leu Thr Thr Glu 115 120 125 Thr Met Leu Gln Ala Glu Glu Arg Ala GlyGly Lys His Gly Asn Lys 130 135 140 Gly Thr Glu Ala Ala Val Thr Ala LeuLys Met Ala Gly Leu Glu Arg 145 150 155 160 Ile 117 227 PRT Arabidopsisthaliana 117 Met Lys Ser Leu Ala Ser Pro Pro Cys Leu Arg Leu Ile Pro ThrAla 1 5 10 15 His Arg Gln Leu Asn Ser Arg Gln Ser Ser Ser Ala Cys TyrIle His 20 25 30 Gly Gly Ser Ser Val Asn Lys Ser Asn Asn Leu Ser Phe SerSer Ser 35 40 45 Thr Ser Gly Phe Ala Ser Pro Leu Ala Val Glu Lys Glu LeuArg Ser 50 55 60 Ser Phe Val Gln Thr Ala Ala Val Arg His Val Thr Gly SerLeu Ile 65 70 75 80 Arg Gly Glu Gly Leu Arg Phe Ala Ile Val Val Ala ArgPhe Asn Glu 85 90 95 Val Val Thr Lys Leu Leu Leu Glu Gly Ala Ile Glu ThrPhe Lys Lys 100 105 110 Tyr Ser Val Arg Glu Glu Asp Ile Glu Val Ile TrpVal Pro Gly Ser 115 120 125 Phe Glu Ile Gly Val Val Ala Gln Asn Leu GlyLys Ser Gly Lys Phe 130 135 140 His Ala Val Leu Cys Ile Gly Ala Val IleArg Gly Asp Thr Thr His 145 150 155 160 Tyr Asp Ala Val Ala Asn Ser AlaAla Ser Gly Val Leu Ser Ala Ser 165 170 175 Ile Asn Ser Gly Val Pro CysIle Phe Gly Val Leu Thr Cys Glu Asp 180 185 190 Met Asp Gln Ala Leu AsnArg Ser Gly Gly Lys Ala Gly Asn Lys Gly 195 200 205 Ala Glu Thr Ala LeuThr Ala Leu Glu Met Ala Ser Leu Phe Glu His 210 215 220 His Leu Lys 225118 141 PRT Methanococcus jannaschii 118 Met Val Leu Met Val Asn Leu GlyPhe Val Ile Ala Glu Phe Asn Arg 1 5 10 15 Asp Ile Thr Tyr Met Met GluLys Val Ala Glu Glu His Ala Glu Phe 20 25 30 Leu Gly Ala Thr Val Lys TyrLys Ile Val Val Pro Gly Val Phe Asp 35 40 45 Met Pro Leu Ala Val Lys LysLeu Leu Glu Lys Asp Asp Val Asp Ala 50 55 60 Val Val Thr Ile Gly Cys ValIle Glu Gly Glu Thr Glu His Asp Glu 65 70 75 80 Ile Val Val His Asn AlaAla Arg Lys Ile Ala Asp Leu Ala Leu Gln 85 90 95 Tyr Asp Lys Pro Val ThrLeu Gly Ile Ser Gly Pro Gly Met Thr Arg 100 105 110 Leu Gln Ala Gln GluArg Val Asp Tyr Gly Lys Arg Ala Val Glu Ala 115 120 125 Ala Val Lys MetVal Lys Arg Leu Lys Ala Leu Glu Glu 130 135 140 119 143 PRTArchaeoglobus fulgidus 119 Met Glu Lys Val Lys Leu Gly Met Val Val AlaGlu Phe Asn Arg Asp 1 5 10 15 Ile Thr Tyr Met Met Glu Ile Leu Gly LysGlu His Ala Glu Phe Leu 20 25 30 Gly Ala Glu Val Ser Glu Val Ile Arg ValPro Gly Thr Phe Asp Ile 35 40 45 Pro Ile Ala Val Lys Lys Met Leu Glu LysGly Arg Val Asp Ala Val 50 55 60 Val Ala Ile Gly Cys Val Ile Glu Gly GluThr Glu His Asp Glu Ile 65 70 75 80 Val Ala Gln His Ala Ala Arg Lys IleMet Asp Leu Ser Leu Glu Tyr 85 90 95 Gly Lys Pro Val Thr Leu Gly Ile SerGly Pro Gly Met Gly Arg Ile 100 105 110 Ala Ala Thr Glu Arg Val Asp TyrAla Lys Arg Ala Val Glu Ala Ala 115 120 125 Val Lys Leu Val Lys Arg LeuLys Glu Tyr Asp Ala Glu Gly Ser 130 135 140 120 139 PRT Methanobacteriumthermoautotrophicum 120 Met Lys Lys Val Arg Ile Gly Ala Val Val Ala GluPhe Asn Tyr Asp 1 5 10 15 Ile Thr His Met Met Leu Glu Leu Ala Lys GluHis Ala Arg Phe Leu 20 25 30 Asp Ala Glu Ile Thr Arg Val Ile Ala Val ProGly Val Phe Asp Met 35 40 45 Pro Leu Ala Val Lys Lys Leu Leu Leu Glu AspGlu Ile Asp Ala Val 50 55 60 Ile Thr Leu Gly Ala Val Ile Glu Gly Ala ThrAsp His Asp Gln Ile 65 70 75 80 Val Val Gln His Ala Ser Arg Lys Ile AlaAsp Leu Ala Leu Asp Tyr 85 90 95 Asp Lys Pro Val Ala Leu Gly Ile Ser GlyPro Gly Met Thr Arg Leu 100 105 110 Glu Ala His Gln Arg Val Asp Tyr AlaLys Arg Ala Val Glu Ala Ala 115 120 125 Val Lys Met Tyr Arg Arg Leu LysGlu Asp Ile 130 135 121 157 PRT Chlamydia trachomatis 121 Met Lys ProLeu Lys Gly Cys Pro Val Ala Lys Asp Val Arg Val Ala 1 5 10 15 Ile ValGly Ser Cys Phe Asn Ser Pro Ile Ala Asp Arg Leu Val Ala 20 25 30 Gly AlaGln Glu Thr Phe Phe Asp Phe Gly Gly Asp Pro Ser Ser Leu 35 40 45 Thr IleVal Arg Val Pro Gly Ala Phe Glu Ile Pro Cys Ala Ile Lys 50 55 60 Lys LeuLeu Ser Thr Ser Gly Gln Phe His Ala Val Val Ala Cys Gly 65 70 75 80 ValLeu Ile Gln Gly Glu Thr Ser His Tyr Glu His Ile Ala Asp Ser 85 90 95 ValAla Ala Gly Val Ser Arg Leu Ser Leu Asp Phe Cys Leu Pro Ile 100 105 110Thr Phe Ser Val Ile Thr Ala Pro Asn Met Glu Ala Ala Trp Glu Arg 115 120125 Ala Gly Ile Lys Gly Pro Asn Leu Gly Ala Ser Gly Met Lys Thr Ala 130135 140 Leu Glu Met Ala Ser Leu Phe Ser Leu Ile Gly Lys Glu 145 150 155122 169 PRT Saccharomyces cerevisiae 122 Met Ala Val Lys Gly Leu Gly LysPro Asp Gln Val Tyr Asp Gly Ser 1 5 10 15 Lys Ile Arg Val Gly Ile IleHis Ala Arg Trp Asn Arg Val Ile Ile 20 25 30 Asp Ala Leu Val Lys Gly AlaIle Glu Arg Met Val Ser Leu Gly Val 35 40 45 Glu Glu Lys Asn Ile Ile IleGlu Thr Val Pro Gly Ser Tyr Glu Leu 50 55 60 Pro Trp Gly Thr Lys Arg PheVal Asp Arg Gln Ala Lys Leu Gly Lys 65 70 75 80 Pro Leu Asp Val Val IlePro Ile Gly Val Leu Ile Lys Gly Ser Thr 85 90 95 Met His Phe Glu Tyr IleSer Asp Ser Thr Thr His Ala Leu Met Asn 100 105 110 Leu Gln Glu Lys ValAsp Met Pro Val Ile Phe Gly Leu Leu Thr Cys 115 120 125 Met Thr Glu GluGln Ala Leu Ala Arg Ala Gly Ile Asp Glu Ala His 130 135 140 Ser Met HisAsn His Gly Glu Asp Trp Gly Ala Ala Ala Val Glu Met 145 150 155 160 AlaVal Lys Phe Gly Lys Asn Ala Phe 165 123 158 PRT Brucella abortus 123 MetAsn Gln Ser Cys Pro Asn Lys Thr Ser Phe Lys Ile Ala Phe Ile 1 5 10 15Gln Ala Arg Trp His Ala Asp Ile Val Asp Glu Ala Arg Lys Ser Phe 20 25 30Val Ala Glu Leu Ala Ala Lys Thr Gly Gly Ser Val Glu Val Glu Ile 35 40 45Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Lys Thr Leu 50 55 60Ala Arg Thr Gly Arg Tyr Ala Ala Ile Val Gly Ala Ala Phe Val Ile 65 70 7580 Asp Gly Gly Ile Tyr Arg His Asp Phe Val Ala Thr Ala Val Ile Asn 85 9095 Gly Met Met Gln Val Gln Leu Glu Thr Glu Val Pro Val Leu Ser Val 100105 110 Val Leu Thr Pro His His Phe His Glu Ser Lys Glu His His Asp Phe115 120 125 Phe His Ala His Phe Lys Val Lys Gly Val Glu Ala Ala His AlaAla 130 135 140 Leu Gln Ile Val Ser Glu Arg Ser Arg Ile Ala Ala Leu Val145 150 155 124 72 DNA Artificial sequence pNCO-N-BS-LuSy sequence 124gaattcatta aagaggagaa attaact atg gcg gcg gcg cgt agc tgc gcg gcc 54 MetAla Ala Ala Arg Ser Cys Ala Ala 1 5 gct atg aat atc ata caa 72 Ala MetAsn Ile Ile Gln 10 15 125 15 PRT Artificial sequence pNCO-N-BS-LuSysequence 125 Met Ala Ala Ala Arg Ser Cys Ala Ala Ala Met Asn Ile Ile Gln1 5 10 15 126 45 DNA Artificial sequence pNCO-C-BS-LuSy sequence 126 cgctca ttt gaa tta gcg gcc gca aac agt tta aaa gga tcc cga 45 Arg Ser PheGlu Leu Ala Ala Ala Asn Ser Leu Lys Gly Ser Arg 1 5 10 15 127 15 PRTArtificial sequence pNCO-C-BS-LuSy sequence 127 Arg Ser Phe Glu Leu AlaAla Ala Asn Ser Leu Lys Gly Ser Arg 1 5 10 15 128 45 DNA Artificialsequence pNCO-BS-LuSy-EC-DHFR sequence 128 cgc tca ttt gaa tta gcg gccgct ggt gga ggc gga atg atc agt 45 Arg Ser Phe Glu Leu Ala Ala Ala GlyGly Gly Gly Met Ile Ser 1 5 10 15 129 15 PRT Artificial sequencepNCO-BS-LuSy-EC-DHFR sequence 129 Arg Ser Phe Glu Leu Ala Ala Ala GlyGly Gly Gly Met Ile Ser 1 5 10 15 130 69 DNA Artificial sequencepNCO-N-VP2-BS-LuSy sequence 130 atg ggg gac ggt gct gtt cag ccg gac ggtggt cag ccg gct gtt cgt 48 Met Gly Asp Gly Ala Val Gln Pro Asp Gly GlyGln Pro Ala Val Arg 1 5 10 15 aac gaa cgt atg aat atc ata 69 Asn Glu ArgMet Asn Ile Ile 20 131 23 PRT Artificial sequence pNCO-N-VP2-BS-LuSysequence 131 Met Gly Asp Gly Ala Val Gln Pro Asp Gly Gly Gln Pro Ala ValArg 1 5 10 15 Asn Glu Arg Met Asn Ile Ile 20 132 74 DNA Artificialsequence pNCO-C-VP2-BS-LuSy sequence 132 cgt tct ttc gaa ggt gac ggt gctgtt cag ccg gac ggt ggt cag ccg 48 Arg Ser Phe Glu Gly Asp Gly Ala ValGln Pro Asp Gly Gly Gln Pro 1 5 10 15 gct gtt cgt aac gaa cgt taggatcc74 Ala Val Arg Asn Glu Arg 20 133 22 PRT Artificial sequencepNCO-C-VP2-BS-LuSy sequence 133 Arg Ser Phe Glu Gly Asp Gly Ala Val GlnPro Asp Gly Gly Gln Pro 1 5 10 15 Ala Val Arg Asn Glu Arg 20 134 69 DNAArtificial sequence pNCO-C-Biotag-BS-LuSy sequence 134 cgt tct ttc gaagcg gcc gca ctc ggc ggc atc ttc gaa gct atg aag 48 Arg Ser Phe Glu AlaAla Ala Leu Gly Gly Ile Phe Glu Ala Met Lys 1 5 10 15 atg gag tgg cgctaaggatcc 69 Met Glu Trp Arg 20 135 20 PRT Artificial sequencepNCO-C-Biotag-BS-LuSy sequence 135 Arg Ser Phe Glu Ala Ala Ala Leu GlyGly Ile Phe Glu Ala Met Lys 1 5 10 15 Met Glu Trp Arg 20 136 54 DNAArtificial sequence pNCO-Lys165-BS-LuSy sequence 136 cgt tct ttc gaa ggtggc ggt ggt tct ggt ggt ggc tct ggt aaa 45 Arg Ser Phe Glu Gly Gly GlyGly Ser Gly Gly Gly Ser Gly Lys 1 5 10 15 taaggatcc 54 137 15 PRTArtificial sequence pNCO-Lys165-BS-LuSy sequence 137 Arg Ser Phe Glu GlyGly Gly Gly Ser Gly Gly Gly Ser Gly Lys 1 5 10 15 138 60 DNA Artificialsequence pNCO-Cys167-BS-LuSy sequence 138 cgt tct ttc gaa ggt ggc ggtggt tct ggt ggt ggc tct ggt ggt ggc 48 Arg Ser Phe Glu Gly Gly Gly GlySer Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 tgc taaggatcc 60 Cys 139 17PRT Artificial sequence pNCO-Cys167-BS-LuSy sequence 139 Arg Ser Phe GluGly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 Cys 140 45 DNAArtificial sequence pFLAG-MAC-BS-LuSy sequence 140 atg gac tac aag gacgac gat gac aaa gtc aag ctt atg aat atc 45 Met Asp Tyr Lys Asp Asp AspAsp Lys Val Lys Leu Met Asn Ile 1 5 10 15 141 15 PRT Artificial sequencepFLAG-MAC-BS-LuSy sequence 141 Met Asp Tyr Lys Asp Asp Asp Asp Lys ValLys Leu Met Asn Ile 1 5 10 15 142 39 DNA Artificial sequencepNCO-C-His6-BS-LuSy sequence 142 cgt tct ttc gaa cat cac cat cac cac cattaaggatcc 39 Arg Ser Phe Glu His His His His His His 1 5 10 143 10 PRTArtificial sequence pNCO-C-His6-BS-LuSy sequence 143 Arg Ser Phe Glu HisHis His His His His 1 5 10 144 69 DNA Artificial sequencepNCO-C-Biotag-AA-LuSy sequence 144 aag tct ctc cga gcg gcc gca ctc ggcggc atc ttc gaa gct atg aag 48 Lys Ser Leu Arg Ala Ala Ala Leu Gly GlyIle Phe Glu Ala Met Lys 1 5 10 15 atg gag tgg cgc taaggatcc 69 Met GluTrp Arg 20 145 20 PRT Artificial sequence pNCO-C-Biotag-AA-LuSy sequence145 Lys Ser Leu Arg Ala Ala Ala Leu Gly Gly Ile Phe Glu Ala Met Lys 1 510 15 Met Glu Trp Arg 20 146 87 DNA Artificial sequencepNCO-His6-C-Biotag-AA-LuSy sequence 146 aag tct ctc cga cat cac cat caccac cat gcg gcc gca ctc ggc ggc 48 Lys Ser Leu Arg His His His His HisHis Ala Ala Ala Leu Gly Gly 1 5 10 15 atc ttc gaa gct atg aag atg gagtgg cgc taaggatcc 87 Ile Phe Glu Ala Met Lys Met Glu Trp Arg 20 25 14726 PRT Artificial sequence pNCO-His6-C-Biotag-AA-LuSy sequence 147 LysSer Leu Arg His His His His His His Ala Ala Ala Leu Gly Gly 1 5 10 15Ile Phe Glu Ala Met Lys Met Glu Trp Arg 20 25 148 99 DNA Artificialsequence pNCO-His6-Gly2-Ser-Gly-C-Biotag-AA-LuSy sequence 148 aag tctctc cga cat cac cat cac cac cat ggc ggt tct ggc gcg gcc 48 Lys Ser LeuArg His His His His His His Gly Gly Ser Gly Ala Ala 1 5 10 15 gca ctcggc ggc atc ttc gaa gct atg aag atg gag tgg cgc taaggatcc 99 Ala Leu GlyGly Ile Phe Glu Ala Met Lys Met Glu Trp Arg 20 25 30 149 30 PRTArtificial sequence pNCO-His6-Gly2-Ser-Gly-C-Biotag-AA-LuSy sequence 149Lys Ser Leu Arg His His His His His His Gly Gly Ser Gly Ala Ala 1 5 1015 Ala Leu Gly Gly Ile Phe Glu Ala Met Lys Met Glu Trp Arg 20 25 30 15045 DNA Artificial sequence pNCO-AA-BglII-LuSy sequence 150 gaattcattaaagaggagaa attaact atg cag atc tac gaa ggt 45 Met Gln Ile Tyr Glu Gly 15 151 6 PRT Artificial sequence pNCO-AA-BglII-LuSy sequence 151 Met GlnIle Tyr Glu Gly 1 5 152 48 DNA Artificial sequencepNCO-AA-BglII-LuSy-(BamHI) sequence 152 aag tct ctc cga gga tcc gtc gacctg cag cca agc tta att agc tga 48 Lys Ser Leu Arg Gly Ser Val Asp LeuGln Pro Ser Leu Ile Ser 1 5 10 15 153 15 PRT Artificial sequencepNCO-AA-BglII-LuSy-(BamHI) sequence 153 Lys Ser Leu Arg Gly Ser Val AspLeu Gln Pro Ser Leu Ile Ser 1 5 10 15 154 11 PRT Artificial sequenceGSVDLQPSLIS-lumazine synthase fusion protein sequence 154 Gly Ser ValAsp Leu Gln Pro Ser Leu Ile Ser 1 5 10

1. Protein conjugate consisting of at least one functional region in anarbitrary position of the sequence of a carrier protein for formation ofa capsid-type spatial structure of the lumazine synthase type, wherebythe outer periphery thereof is covalently linked with a multiple numberof the functional regions.
 2. Protein conjugate which can be produced byrecombinant technology and which consists of at least one functionalprotein region at the N-terminus and/or C-terminus and/or inserted intoa loop region of the sequence of a carrier protein region for formationof a capsid-type spatial structure of the lumazine synthase type,whereby the outer periphery thereof is covalently linked with a multiplenumber of the functional regions.
 3. Protein conjugate according toclaim 1 and 2, whereby the carrier protein region comprises an aminoacid sequence—selected from a set of sequences—which is obtained by aprocedure whereby for every amino acid position in the sequence of apredetermined native lumazine synthase, an amino acid or a deletion isselected from the respective position of an alignment of thepredetermined lumazine synthase sequence with at least one nativelumazine synthase sequence of another organism.
 4. Protein conjugateaccording to claim 1 and 2, whereby the carrier protein region has thesequence of a native lumazine synthase.
 5. Protein conjugate accordingto claim 1 and 2, whereby the carrier protein region has the sequence ofa thermostable native lumazine synthase.
 6. Protein conjugate accordingto claim 1 and 2 whereby the thermostable native lumazine synthase hasthe protein sequence of the lumazine synthase of a hyperthermophilicmicroorganism, preferentially Aquifex aeolicus.
 7. Protein conjugateaccording to claim 1 and 2 whereby the carrier protein region consistsof a mixed sequence comprising amino acid positions 1-60 of the nativelumazine synthase of a mesophilic organism referenced to Bacillussubtilis, and the amino acid positions 61-154 of the native lumazinesynthase of a hyperthermophilic microorganism referenced to Aquifexaeolicus.
 8. Protein conjugate according to claim 1 and 2, whereby thecarrier protein region consists of an arbitrary sequence, whereby themain chain of the sequence folds into α-helix and β-pleated sheetmotifs, whereby 4 β-segments form a parallel 4-stranded β-pleated sheetwhich is flanked on both sides by two respective α-helices, whereby 5units of these α-β-motifs associate under formation of a pentamericstructure, whereby the N-terminus of each unit can form the fifthβ-segment to the central 4-stranded β-pleated sheet of the adjacentunit, whereby 12 of these pentameric substructures associate underformation of the icosahedral structure of a lumazine synthase andwhereby the N- and C-termini of the arbitrary sequence with thestructural characteristics described above are located at the surface ofthe hereby formed icosahedron and whereby the arbitrary sequence ispreferentially obtained by the comparison of a set of sequences ofdifferent lumazine synthase sequences, i.e. lumazine synthase sequencesderived from lumazine synthase genes of different organisms, inparticular by search algorithms according to Altschul et al. (1997). 9.Protein conjugate according to claim 1 and 2 whereby the carrier proteinregion comprises a sequence of a native lumazine synthase whereby atleast one cystein unit is replaced by another amino acid or is deletedor is chemically modified.
 10. Protein conjugate according to claim 9whereby a cystein unit in a position corresponding to one of thepositions 93 and/or position 139 of the lumazine synthase of Bacillussubtilis is deleted or is replaced by another aminoacid, preferentiallyserine.
 11. Protein conjugate according to one of the claims 1 to 10,whereby the carrier protein region and the functional protein region arelinked by a linker peptide.
 12. Protein conjugate according to one ofthe claims 2 to 11, whereby the functional protein region is thesequence of a dihydrofolate reductase, a maltose binding protein, aprotein that is susceptible to in vivo biotinylation, an antigenicallyactive peptide, especially from a surface protein of a virus, a peptidethat can be recognized by a monoclonal antibody, a stochasticallygenerated peptide or an amino acid that is susceptible to chemicalderivatization, e.g. cystein or lysin.
 13. Protein conjugate accordingto one of the claims 2 to 12, whereby the carrier protein region ischemically modified.
 14. Protein conjugate according to one of theclaims 2 to 13, whereby the functional protein region is chemicallymodified, preferably biotinylated.
 15. Heterooligomeric proteinconjugate consisting of mixtures of at least two different proteinconjugates according to one of the claims 1 to 14 or at least oneprotein conjugate according to one of the claims 1 to 14 and at leastone carrier protein region without functional protein region with asequence according to one of the claims 3 to 8, whereby the individualproteins are covalently coupled by chemical treatment if required. 16.Procedure for preparation of a protein conjugate according to claim 1characterized by the following steps, a) isolation of a lumazinesynthase from a wild type or a recombinant organism (carrier protein);b) chemical coupling of functional molecules to the carrier protein. c)purification of the protein conjugate.
 17. Procedure for preparation ofa protein conjugate or a heterooligomeric protein according to one ofthe claims 2 to 14 which is characterized by the following steps, a)Preparation of a first DNA coding for the carrier protein region b)Fusion of at least one second DNA coding for the functional region andfor the linker protein, if required, at the 5′ end and/or the 3′ end ofthe first DNA and/or insertion of the second DNA into a region of thefirst DNA coding for a loop region of the carrier protein underformation of an artificial DNA. c) Conversion of the artificial DNA ofstep b) into an expression plasmid. d) Transformation of host cells withone or several of the expression plasmids generated in step c). e)Expression of the artificial DNA in the transformed host cells underformation of a protein conjugate, if required under introduction of apredetermined post-translational modification of the protein conjugatein vivo, preferably by phosphorylation, glycosidation or biotinylation.f) Purification of the protein conjugate. g) Modification of the proteinconjugate, if required, by chemical coupling of amino acid residues onthe protein surface of a capsid-type spatial structure formed from theprotein conjugate with arbitrarily determined coupling partners. 18.Procedure according to claim 15 characterized by the production of aheterooligomeric protein by a) mixing of different protein conjugatesobtained according to claim 16, step c) and/or claim 17, step f) b)denaturation of the resulting mixture and c) renaturation of themixture; or by a₂) denaturation of different protein conjugates obtainedaccording to claim 16, step c) and/or claim 17, step f) b₂) mixing thedenatured protein conjugate c₂) renaturation of the mixture 19.Procedure according to claim 15, characterized by the use of proteinconjugates which were produced with the use of a ligand which supportsthe folding
 20. Vectors for preparation of the protein conjugatesaccording to one of the claims of 2 to
 14. 21. DNA coding for a proteinaccording to claim
 20. 22. Protein consisting of the lumazine synthaseof Bacillus subtilis, whereby the amino acid cystein in position 93 isreplaced by the amino acid serine.
 23. Protein consisting of thelumazine synthase of Bacillus subtilis whereby the amino acid cystein inposition 139 is replaced by the amino acid serine.
 24. Protein,consisting of the lumazine synthase of Bacillus subtilis whereby theamino acid cystein in the positions 93 and 139 is replaced by the aminoacid serine.
 25. DNA adapted to the codon usage of Escherichia coli forpreparation of the lumazine synthase of Aquifex aeolicus in arecombinant Escherichia coli strain.
 26. Protein consisting of thelumazine synthase of Aquifex aeolicus for use as carrier proteinaccording to claim
 1. 27. Chimeric protein consisting of the amino acids1-60 of the lumazine synthase of Bacillus subtilis and the amino acids61-154 of the lumazine synthase of Aquifex aeolicus for use as carrierprotein according to claim
 1. 28. Vector for preparation of proteinconjugates according to claim 12, whereby the functional DNA part islocated at the 5′ end of the carrier protein gene of the lumazinesynthase type, whereby the fused gene codes for an artificial proteinwhich contains a functional protein region, a carrier protein region forformation of a capsid-type spatial structure of the lumazine synthasetype and optionally a linker peptide, and whereby the functional proteinregion and the linker peptide are located at the N-terminus of thecarrier protein region and whereby the vector contains the followingcomponents: a) a DNA fragment coding for a carrier protein region forformation of a capsid type spatial structure of the lumazine synthasetype b) a DNA fragment coding for an arbitrarily selected functionalprotein region. c) optional: a DNA fragment coding for a linker peptide.29. A vector according to claim 28 whereby it contains the gene for thelumazine synthase of Bacillus subtilis coding for the carrier proteinregion, the gene for the dihydrofolate reductase of Escherichia colicoding for the functional protein region and, as linker peptide, a DNAfragment coding for a tripeptide consisting of the amino acid alanine.30. A vector according to claim 28 whereby it contains the gene for thelumazine synthase of Bacillus subtilis coding for the carrier proteinregion, the gene for the “maltose binding protein” of Escherichia colicoding for the functional protein region and as linker peptide a DNAfragment coding for the amino acid sequence SNNNNNNNNNNLGIEGRISEFAAA.31. Vector for preparation of protein conjugates according to claim 12,whereby the functional DNA part is located at the 3′ end of the carrierprotein gene of the lumazine synthase type and whereby the fused genecodes for an artificial protein which contains a functional proteinregion, a carrier protein region for formation of a capsid-type spatialstructure of the lumazine synthase type, and optionally a linkerpeptide, and whereby the functional protein region and the linkerpeptide are located at the C-terminus of the carrier protein region andwhereby the vector contains the following components: a) a DNA fragmentcoding for a carrier protein region for formation of a capsid-typespatial structure of the lumazine synthase type (without respective stopcodon) b) a DNA fragment coding for an arbitrarily selected functionalprotein region c) optional: a DNA fragment coding for a linker peptide.32. Vector according to claim 31 whereby it contains the gene for thelumazine synthase of Bacillus subtilis coding for the carrier proteinregion, the gene for dihydrofolate reductase of Escherichia coli codingfor the functional protein region and as linker peptide a DNA fragmentcoding for the amino acid sequence LAAAGGGG.
 33. Vector according toclaim 31 whereby it contains the gene for the lumazine synthase ofAquifex aeolicus (according to claim 25) coding for the carrier proteinregion and a gene fragment coding for the functional protein region withthe amino acid sequence GSVDLQPSLIS. The vector comprises a singularrecognition sequence at the 5′ end of the gene sequence of the carrierprotein for the restriction endonucleases BglII, whereby thisrestriction site can be used for the fusion of foreign genes to the 5′end of the lumazine synthase.
 34. Vector for preparation of proteinconjugates according to claim 12, whereby the functional DNA part islocated at the 3′ end of the carrier protein gene of the lumazinesynthase type and whereby the fused gene codes for an artificial proteinwhich contains a functional protein region, a carrier protein region forformation of a capsid-type spatial structure of the lumazine synthasetype, and optinally a linker peptide, and whereby the functional proteinregion and the linker peptide are located at the C-terminus of thecarrier protein region and whereby the selected functional proteinregion is biotinylated in vivo and whereby the vector contains thefollowing components: a) a DNA fragment coding for a carrier proteinregion for formation of a capsid-type spatial structure of the lumazinesynthase type (without respective stop codon) b) a DNA fragment codingfor a peptide susceptible to biotinylation with the sequenceLGGIFEAMKMEWR, whereby the amino acid lysin is biotinylated in vivo c)optional: a DNA fragment coding for a linker peptide.
 35. A vectoraccording to claim 34 whereby it contains the gene for the lumazinesynthase of Bacillus subtilis coding for the carrier protein region andas linker peptide a DNA fragment coding for a tripeptide consisting ofthe amino acid alanine.
 36. A vector according to claim 34 whereby itcontains the gene, adapted to the codon usage of Escherichia coli,coding for the lumazine synthase of Aquifex aeolicus according to claim25 as carrier protein region and as linker peptide a DNA fragment codingfor a tripeptide consisting of the amino acid alanine.
 37. A vectoraccording to claim 34 whereby it contains the gene, adapted to the codonusage of Escherichia coli, coding for the lumazine synthase of Aquifexaeolicus according to claim 25 as carrier protein region and as linkerpeptide a DNA fragment coding for a peptide consisting of the amino acidsequence HHHAAA.
 38. A vector according to claim 34 whereby it containsthe gene adapted to the codon usage of Escherichia coli coding for thelumazine synthase of Aquifex aeolicus according to claim 25 as carrierprotein region and as linker peptide a DNA fragment coding for a peptideconsisting of the amino acid sequence HHHHHHGGSGAAA.
 39. Vector forproduction of a protein conjugate according to claim 12 whereby thefunctional DNA part is located at the 5′ end of the lumazine synthasegene of Bacillus subtilis, whereby the functional DNA part codes for anantigenically active peptide of the VP2 surface protein of the “minkenteritis virus” and the fused gene codes for an artificial proteincomprising a functional protein part and a carrier protein part andwhereby the functional protein part is located at the N-terminus of thelumazine synthase and whereby the vector has the following components:a) Lumazine synthase gene of Bacillus subtilis. b) DNA at the 5′ end ofthe lumazine synthase gene coding for peptide. The foreign peptide hasthe sequence MGDGAVQPDGGQPAVRNER.
 40. Vector for production of a proteinconjugate according to claim 12 whereby the functional DNA part islocated at the 3′ end of the lumazine synthase gene of Bacillussubtilis, whereby the functional DNA part codes for an antigenicallyactive peptide from the VP2 surface protein of the “mink enteritisvirus”, and whereby the fused gene codes for an artificial proteincomprising a functional protein part and a carrier protein part, wherebythe functional protein part is located at the C-terminus of the lumazinesynthase and whereby the vector contains the following components: a)Lumazine synthase gene from Bacillus subtilis (without stop codon). b)DNA coding for peptide at the 3′ end of the lumazine synthase gene. Theforeign peptide has the sequence GDGAVQPDGGQPAVRNER.
 41. Vector forproduction of a protein conjugate according to claim 12 whereby thefunctional DNA part is located at the 5′ end and at the 3′ end of thelumazine synthase gene of Bacillus subtilis, and whereby the functionalDNA part codes for an antigenically active peptide from the VP2 surfaceprotein of the “mink enteritis virus”, and whereby the fused gene codesfor an artificial protein comprising a functional protein part and acarrier protein part, and whereby the functional protein part is locatedat the N-terminus as well as at the C-terminus of the lumazine synthaseand whereby the vector contains the following components: a) Lumazinesynthase gene from Bacillus subtilis (without stop codon). b) Twosequences coding for peptides at the 5′ and the 3′ end of the lumazinesynthase gene. The peptide at the N-terminus has the sequenceMGDGAVQPDGGQPAVRNER, the peptide at the C-terminus has the sequenceGDGAVQPDGGQPAVRNER.
 42. Vector for production of a protein conjugateaccording to claim 12 whereby the functional DNA region is located atthe 5′ end of the lumazine synthase gene from Bacillus subtilis and thefunctional DNA part codes for an octapeptide (FLAG peptide) which isrecognized by a monoclonal antibody (preferentially Anti-FLAG-M2; IBI E.coli FLAG® Expression System, Integra Biosciences, Fernwald), wherebythe fused gene codes for an artificial protein which contains afunctional protein region and a carrier protein region and whereby thefunctional protein region is located at the N-terminus of the lumazinesynthase and whereby the vector comprises the following components: a)Lumazine synthase gene from Bacillus subtilis b) DNA coding for peptideat the 5′ end of the lumazine synthase gene. The foreign peptide has thesequence MDYKDDDDK; c) DNA coding for a linker peptide with the sequenceVKL
 43. Vector for production of a protein conjugate according to claim12 whereby the functional DNA region is located at the 3′ end of thelumazine synthase of Bacillus subtilis, whereby the functional DNA partcodes for a hexapeptide (His6-peptide) which is recognized by amonoclonal antibody (preferentially Penta-His™ antibody; Qiagen, Hilden)and whereby the fused gene codes for an artificial protein comprising afunctional protein region and a carrier protein region and whereby thefunctional protein region is located at the C-terminus of the lumazinesynthase and whereby the vector contains the following components: a)Lumazine synthase gene from Bacillus subtilis without stop codon. b) DNAcoding for peptide at the 3′ end of the lumazine synthase gene. Theforeign peptide has the sequence HHHHHH.
 44. Vector for production of aprotein conjugate according to claim 12 whereby the functional DNAregion is located at the 3′ and of the lumazine synthase gene fromBacillus subtilis, whereby the functional DNA region codes for anartificial peptide sequence which ends with the amino acid lysin,whereby the amino acid lysin can be used for chemical coupling offunctional molecules to the carrier protein region (cf. claim 16 b) andwhereby the functional protein region serves as linker (tentacle-linker)and whereby the fused gene codes for an artificial protein comprising afunctional protein region and a carrier protein region, whereby thefunctional protein region is located at the C-terminus of the carrierprotein region and whereby the vector comprises the followingcomponents: a) Lumazine synthase gene from Bacillus subtilis (withoutstop codon). b) Codon for lysin (aaa) at the 3′ end of the artificialDNA. c) DNA coding for a linker peptide with the sequence GGGGSGGGSG.45. Vector for production of a protein conjugate according to claim 12whereby the functional DNA region is located at the 3′ end of thelumazine synthase gene from Bacillus subtilis, whereby the functionalDNA region codes for an artificial peptide sequence which ends with theamino acid cystein, whereby the amino acid cystein can be used forchemical coupling of fusion molecules to the carrier protein region (cf.claim 16 b), and whereby the functional protein region serves as linker(tentacle linker) and whereby the fused gene codes for an artificialprotein which comprises a functional protein region and a carrierprotein region, whereby the functional protein region is located at theC-terminus of the carrier protein region and whereby the vectorcomprises the following components: a) Lumazine synthase gene fromBacillus subtilis (without stop codon). b) Codon for cystein (tgc) atthe 3′ end of the artificial DNA. c) DNA coding for a linker peptidewith the sequence GGGGSGGGSGGG.
 46. Protein conjugates according to oneof the claims 1 to 15 for preparation of a medicament (pharmacologicalagent) or a vaccine.
 47. Use of the protein conjugates according to oneof the claims 1 to 15 for preparation of a medicament (pharmacologicalagent) or a vaccine.
 48. Use of the protein conjugates according to oneof the claims 1 to 15 for preparation of diagnostically ortherapeutically applicable antibodies
 49. Use of the protein conjugatesaccording to one of the claims 1 to 15 for selective detection ofantibodies or for purification of antibody mixtures or forcharacterization of antibodies.
 50. Use of protein conjugates accordingto one of the claims 1 to 15 for preparation of protein libraries 51.Medicaments (pharmacological agents) containing a pharmacologicallyactive quantity of a protein conjugate according to one of the claims 1to 15
 52. Vaccine containing an immunologically active quantity of aprotein conjugate according to one of the claims 1 to 15
 53. Use ofprotein conjugates according to one of the claims 1 to 15 as biosensor.